CN109982715A

CN109982715A - Using low dosage volume B cell epitope composition in the method for the induction of antibodies immune response in the experimenter

Info

Publication number: CN109982715A
Application number: CN201680091084.1A
Authority: CN
Inventors: M·曼索; F·奥斯; M·斯坦福德; L·萨玛特; R·拉迦格帕拉; L·D·马克德纳德
Original assignee: Immunovaccine Technologies Inc
Current assignee: Immunovaccine Technologies Inc
Priority date: 2016-09-27
Filing date: 2016-09-27
Publication date: 2019-07-05
Also published as: US20220347296A1; JP2022116014A; JP2019532943A; EP3518963A4; WO2018058230A1; EP3518963A1; JP7125197B2; CA3038155A1; JP7452931B2; US11406705B2; AU2016425290A1; US20190224312A1

Abstract

Present disclose provides for the method for induction of antibodies immune response, application and composition and kit in the experimenter.Methods and applications are related to parenterally giving the composition of low dosage volume, the composition includes antigen, amphipathic compound and hydrophobic carrier comprising B cell epitope, and wherein the low dosage volume of composition induces the antibody mediated immunity response to B cell epitope less than 100 μ l and in the experimenter.

Description

Using low dosage volume B cell epitope composition in the experimenter induction of antibodies exempt from The method of epidemic disease response

Technical field

It induces and resists in the experimenter the present invention generally relates to the composition for giving low dosage volume by parenteral The method of body immune response, the composition include the antigen containing B cell epitope, amphipathic compound and hydrophobic carrier.

Background technique

B cell epitope is the segment of the protein for example identified by the antibody generated by B cell.Continuity B cell table Position, such as the linear segments (segment) of protein, can be used as antigen in subunit vaccine with induction of antibodies response.However, even Continuous property B cell epitope has had limited success in exploitation.This may be partly due to antigen to the invalid of immune system Delivering.

In the vaccine that Canada ratifies at present, according to disease controls in 2016 and prevention center (Centers for Disease Control and Prevention) (CDC) for 65 years old or more adult being given by intramuscular route Suggestion (CDC website-is immune to practice Advisory Board (ACIP) vaccine suggestion (Advisory Committee for Immunization Practices (ACIP) Vaccine Recommendations)), the range of dose volume be 0.5mL extremely 1.0mL.Quadrivalent (Fluzone Intradermal Quadrivalent) (Sanofi intradermal for Fluzone Pasteur), the minimum dose volume of the vaccine of approval is 0.1mL；However, only for the adult between 18-64 years old and passing through skin Interior approach is given.

In the disclosure, we report is induced in the experimenter using the composition that the parenteral of low dosage volume is given The new method of antibody mediated immunity response, the composition include the antigen containing B cell epitope, amphipathic compound and hydrophobic carrier.

Summary of the invention

In one embodiment, this disclosure relates in the experimenter induction of antibodies immune response method, including The composition of low dosage volume is given to experimenter's parenteral, the composition includes: the antigen comprising B cell epitope；Amphiphilic Close object；And hydrophobic carrier, wherein the low dosage volume of composition is induced less than 100 μ l and in the experimenter to B cell epitope Antibody mediated immunity response.

In one embodiment, this disclosure relates to which the composition of low dosage volume is thin to B for inducing in the experimenter The application of the antibody mediated immunity response of born of the same parents' epitope, the composition include: the antigen comprising B cell epitope；Amphipathic compound；And it is hydrophobic Carrier, wherein composition is given for parenteral, and the low dosage volume of composition is less than 100 μ l.

It is about for the low dosage volume that parenteral is given in method disclosed herein and an embodiment of application 50μl.In one embodiment, it is intramuscular injection that parenteral, which is given,.

In method disclosed herein and an embodiment of application, antigen includes RSV plants of A subgroup people or B subgroup people The extracellular domain of RSV plants of small hydrophobin (SH) or small hydrophobin by RSV plants of RSV plants of A subgroup people or B subgroup people (SH) extracellular domain is constituted.In one embodiment, antigen includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1), NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) or Its segment, or by amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1), NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) or its segment are constituted.

In method disclosed herein and an embodiment of application, amphipathic compound is lipid or lipid mixture. In one embodiment, amphipathic compound is the lipid mixture of dioleyl phosphatidyl choline (DOPC) and cholesterol.

In method disclosed herein and an embodiment of application, hydrophobic carrier is mineral oil or mannide The mineral oil solution of oleate (mannideoleate), such asISA 51 VG.

In method disclosed herein and an embodiment of application, composition includes: containing amino acid sequence The peptide of NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1)；Contain dioleyl phosphatidyl choline (DOPC) and cholesterol Lipid mixture；Short synthesis lipopeptid, is PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)；And ISA 51 VG。

In method disclosed herein and an embodiment of application, composition is free from water or generally not aqueous.

After combining appended claims and attached drawing commentary to be described below, other aspects of the invention, embodiment and spy Sign will be apparent those of ordinary skill in the art.

Detailed description of the invention

Only pass through example example embodiments of the present invention in the figure:

Fig. 1 shows the resisting for RSV SHe A peptide antigen detected in the serum from subject in low peptide group The level of body.Show the result of the 28th (A) of research, 56 (B), 84 (C) and 236 days (D).Subject in oil base group is grinding Study carefully and receives within the 0th day and the 56th day to be inoculated with twice.Subject in alum base (alum-based) group received primary at research the 0th day It is inoculated with and in the research placebo of receiving in the 56th day.Subject in placebo received at research the 0th day and the 56th day Placebo injection.Result lower than detection limit is reported as log10=0.Data (n=4- is shown for each independent subject 8), lines indicate average value.Pass through student ' s t inspection to compare oil-based formulation with alum based formulation to carry out computational statistics significant Property:^*P < 0.05,^***p<0.001.

Fig. 2 shows detected in the serum from subject in high peptide group for the anti-of RSV SHe A peptide antigen The level of body.Show the result of the 28th (A) of research, 56 (B) and the 84th day (C).Subject in oil base group is in research the 0th It and receiving in the 56th day are inoculated with twice.Subject in alum base group received primary inoculation at research the 0th day and in research the The placebo of receiving in 56 days.Subject in placebo receives placebo injection in the 0th day and the 56th day in research.Lower than inspection The result for surveying limit is reported as log10=0.Data (n=4-8) is shown for each independent subject, and lines indicate average value. Pass through that oil-based formulation is compared in student ' s t inspection and alum based formulation carrys out computational statistics conspicuousness:^*P < 0.05,^***P< 0.001.

Fig. 3 is shown through not add (non-adjuvanted) the oil base vaccine of adjuvant, add adjuvant (adjuvanted) after the RSV SHe A peptide antigen and RSV SHe B peptide antigen inoculation of oil base vaccine or water base vaccine formulation RSV SHe A and RSV the SHe B antibody titer induced in mouse.At research the 0th day and the 28th day to the mouse in each group It is inoculated with and every four weeks carries out bloodletting for the Serum Antibody Detection by ELISA.It is examined by unidirectional ANOVA through Tukey post The significance,statistical tested:^**P < 0.01,^***p<0.001。

Specific embodiment

For the synthesizing linear B cell epitope induction given, strong and effective antibody mediated immunity response is tired in the experimenter Difficult and usually need repeatedly immune, wherein typical doses volume is 0.5mL to 1.0mL.Nonetheless, using thin comprising B Being immunized for the composition of born of the same parents' epitope is usually unsuccessful in the experimenter, wherein preclinical study excessively prediction in the mankind at Function.Limited success has been obtained at continuity B cell epitope in fact, developing vaccine composition.

In the past, we have demonstrated that the combination that dose volume is the DepoVax base vaccine and cyclophosphamide of 0.1mL can be used for Cause to the cellullar immunologic response of peptide antigen (by CD8⁺T cell mediates).However, being related to conjoint therapy and different siberian crabapples These discoveries of system aspect (arm) (cellullar immunologic response), which do not provide, is directed to B cell using even lower dose volume to induce Any introduction or suggestion of the method for disclosure of the humoral immune response (being mediated by B cell) of epitope.

The disclosure provides the method for the induction of antibodies immune response in the experimenter.Surprisingly, the present inventor is It has been observed that the composition comprising the antigen containing B cell epitope, amphipathic compound and hydrophobic carrier can be with low dosage volume stomach It gives outside and strong and effective antibody mediated immunity response is provided in the experimenter.The result obtains in people's clinical test It proves.

As shown in the example of this paper, the method disclosed herein using low dosage volume can be respectively at the 236th day and 84 days in 100% subject for being inoculated with low peptide disclosed herein (10 μ g antigen) or high peptide (25 μ g antigen) oil-based composition Inducing antigen-specific antibody mediated immunity response (Fig. 1 and 2).The dose volume of composition is only 50 μ l.In this low dosage volume Under, surprisingly, the experimenter is fully able to generate antibody mediated immunity response, needless to say reaches this 100% subject's Degree.

In addition, the 56th day the results show that method disclosed herein can be inoculated with respectively low peptide (10 as described herein μ g antigen) and 50% and 62.5% subject of high peptide (25 μ g antigen) oil-based composition in (Fig. 1 and 2) inducing antigen-specific Antibody mediated immunity response.In contrast, the experimenter for being inoculated with aqueous alum composition does not induce detectable resist generally completely Body immune response, wherein only 25% low peptide subject and 12.5% high peptide subject show detectable antibody mediated immunity and answer It answers.Therefore, compared with alum composition, the quantity of the subject with detectable antibody mediated immunity response is as described herein low In the case where peptide combinations high 2.0 times and it is 5.0 times high in the case where high peptide combinations as described herein.

Except the quantity increase for showing the experimenter of detectable antibody mediated immunity response except through method disclosed herein, The antibody titer obtained using disclosed method and composition is also significant higher --- compared with using aqueous alum composition. As shown in table 1, mean end point titre (endpoint in the case where low peptide (10 μ g antigen) oil-based composition as described herein Titer) high by about 16.7 at the 56th day at the 28th day high about 123.2 times --- compared in the case where alum composition.It is right In high peptide (25 μ g antigen) oil-based composition as described herein, mean end point titre is at the 28th day high about 35.3 times and at the 56th day It is about 13.1 times high --- compared in the case where alum composition.Only it is single give composition in the case where just observe this The increase of kind antibody titer.This enhancing level in the experimenter is astonishing and unexpected.

Result in example further proves, when method disclosed herein can be at least as early as 28 days after single give Strong antibody mediated immunity response is induced in the experimenter, and proves the antibody mediated immunity response sustainable extended period.For example, In the case where only giving (the 0th day and the 56th day) twice, high-caliber antibody titer still continues at least up to the 84th day and the 236 days.(Fig. 1 and 2；Table 1).Disclosed method induction of antibodies immune response --- it is only low dosage volume gives group twice Close the lasting time span in the case where object --- ability be surprising result.

In general, the results show that the aqueous alum composition of low dosage volume induces in the sufficient amount of experimenter It is generally invalid in terms of strong antibody mediated immunity response.Surprisingly however it was, low dosage volume is thin comprising containing B The composition of the antigen of born of the same parents' epitope, amphipathic compound and hydrophobic carrier is in most of (low peptides) or all (high peptide) experimenters Induce strong antibody mediated immunity response.Without being bound by theory, this may be due to one of following or a variety of: potentially solve It is related to the nanoparticulate drug delivery system of the deliquescent some formulation problems of antigen；The controlled release of compositions disclosed herein and Slow release characteristic；Overcome the bioavailability of difference；Lesser drug dose；Reduce administration variability；Increase in the anti-of injection site Former stability；Increase the resistance etc. to nonspecific degradation.

Clinical study results are also shown, and the tolerance of the composition of low dosage volume disclosed herein is good and have can The safety (safety profile) of receiving.As used herein, from " acceptable safety ", at least mean not appoint The report of what serious adverse events (adverse event).As described in example 1 above, it is not reported in all research participants Accuse serious adverse events.

As used herein, " serious adverse events " (SAE) be cause it is dead, be threat to life (that is, when event occurs Have mortality risk), need to be hospitalized or after extending the existing hospital stays, leading to disability/incompetence or study subject The unfortunate medical events of congenital abnormality/birth defect (medical occurrence) in generation.

As discussed in embodiment 1, the local adverse events of (solicited) of the most common demand of report are slightly to infuse Penetrate position pain.It is believed that this injection site reaction level will not need any kind of pain management (pain therapy, pain management).In addition, injection site pain is not observed in the case where the delivering of the oil-based composition of booster Increase.

The whole body adverse events of the most common demand of report are drowsiness, nausea, diarrhea and myalgia.For overall non-demand Adverse events, unreported there are serious adverse events, and restore or solve all other event.

As used herein, term " adverse events of demand " is preassigned result --- the participant in clinical test Be required record such as whether in the presence of, and if it is present with apply certain strength grade.The 0th after giving research vaccine Its daily adverse events for collecting demand to the 6th day.Following part (injection site) adverse events are demands: at injection site Pain；Injection site rubescent (redness) and injection site swelling.Following whole body (whole body (systemic)) adverse events are Demand: drowsiness；Fever；Nausea；Diarrhea；Vomiting；With systemic myalgia.

As used herein, the event of non-demand is to inquire with non-style of leadership (non-leading manner) from last The event of participant's confirmation when their health whether there is any variation since study visit in clinical research.In epidemic disease Seedling give after study visit during collect the adverse events of non-demand.

It is as described herein the result is that surprising and unexpected --- at least because of known comprising B cell epitope Vaccine composition does not usually work completely for generating detectable antibody mediated immunity response in the experimenter.Moreover, recognizing in the past For bigger volume, (such as 0.5mL to 1.0mL) is significant to composition (for example, intramuscular) is parenterally given (significant), it is spread in order to provide sufficient whole body effectively to engage (engage) Immune System Components (as aqueous Vaccine) and/or Immune System Components are attracted to injection site (as oil base vaccine).

Method disclosed herein and the embodiment of composition will be more fully described in following part.

Method for induction of antibodies response

In one embodiment, this disclosure relates in the experimenter induction of antibodies immune response method, including The composition of low dosage volume is parenterally given to the experimenter, the composition includes: the antigen comprising B cell epitope；Amphiphilic Compound；And hydrophobic carrier, wherein the low dosage volume of composition is induced less than 100 μ l and in the experimenter to B cell table The antibody mediated immunity response of position.

As used herein, " induction " or " with induction " antibody mediated immunity response is generation, initiation, improvement and/or booster immunization Response.From " generation " or " initiation ", mean that the experimenter was not developed in the past, and/or without the anti-of the progress for antigen Body immune response, and method disclosed herein leads to detectable antibody mediated immunity response in subject.From " improvement " or " add It sees by force ", means that antibody mediated immunity is answered relative to previous state of immune response (for example, before application method of the invention) It answers enhanced, promotion or strengthens so that subject is benefited.

The term " detectable " in the context of " detectable antibody mediated immunity response " means antigen spy as used herein Heterogenetic antibody immune response can detect in the experimenter, such as example pass through body fluid (bodily fluid) (such as whole blood, serum Deng) analysis or assessed by therapeutic treatment to subject.As illustrative embodiments, " detectable antibody mediated immunity is answered Answer " it is such detectable antibody mediated immunity response, wherein can be inhaled under the detection limit of 1/100 dilution by enzyme linked immunological Attached measurement (ELISA) detects antibody titer in the human serum sample from immunized subject, as described in present example.

In some embodiments, " induction " or " to induce " means to answer in the antibody mediated immunity for causing or generating for antigen Answer the effect of middle presence improves.As used herein, " the effect of improvement ", " improving effect " etc., refer to the immune response of subject Composition can be made in treatment disease or illness fermentation more effectively any variation or change.In some embodiments, this can It is related to accelerating the appearance of immune response and/or improves the persistence or intensity of immune response.

In some embodiments, " induction " or " with induction " refers in the past by early immune or for its of antigen It, which is exposed, generates, causes, strengthens or extends antigentic specificity anamnedstic response in (primed) subject for having contacted antigen Ability.The contact with occurring when being exposed to antigen for the first time antigen immune response on the contrary, recall immune response be such Immune response, when occurring at second and/or being subsequently exposed to antigen, by primary immune or for antigen before rebuilding The immune response that other exposures (such as previous pathogenic infection) generate.

In some embodiments, " induction " or " reinforcement " refers in the past by early immune or for the other of antigen Exposure had contacted the ability that (boost) antigen-specific antibodies immune response is maintained and/or reinforced in the subject of antigen.From " maintain and/or reinforce " sees, means, promotion enhanced with the immune response of pre-induction, improves, strengthens or extend so that tested Person is benefited.

As used herein, " reinforcement " includes the case where such, wherein keep the antibody mediated immunity response for antigen more effective or Person is in intensity and/or adverse events are avoided, eliminated or mitigated on the duration.From " more effective ", mean relative to tested The previous state of immune response of person, enhancing are promoted, are improved, strengthened or extending immune response so that subject is benefited.Some In embodiment, " reinforcement " refers to the ability for reducing the generation of adverse events.For example, in one embodiment, booster immunization Response may include the injection site reaction as caused by the composition or different components as described herein of biggish dose volume The reduction of generation.

Method disclosed herein is related to parenterally giving the application of the composition of " low dosage volume " of the experimenter.Such as this Text uses, and " low dosage volume " refers to the total volume that the composition of the experimenter is given in single give.From " single to give ", It means all dosage (complete dose) in particular point in time (such as the 0th day) by single parenteral injection or infusion Give subject.

" low dosage volume " is less than the composition as described herein of 100 μ l.In some embodiments, low dosage volume It is the group of about 50 μ l, about 55 μ l, about 60 μ l, about 65 μ l, about 70 μ l, about 75 μ l, about 80 μ l, about 85 μ l, about 90 μ l or about 95 μ l Close object.In some embodiments, low dosage volume is in about 50 μ l to the composition between about 75 μ l.In some embodiments In, low dosage volume is about 50 μ l or exactly 50 μ l.Low dosage volume be can in the experimenter induction of antibodies immune response Volume.

It compares with " low dosage volume ", term " dosage " (dose) as used herein " refer to included in low dosage volume In vaccine component (as such as antigen) total amount or amount (total amount or quantity) (such as microgram, milligram, rub Your amount etc.).Term " administration (dose) " can also be used interchangeably with " giving " herein, be combined with indicating to give to the experimenter The action of object.

From " parenteral is given " or " parenterally giving ", mean by injecting or to give composition by infusion. In some embodiments, give approach can be intravenous, intramuscular, subcutaneous, peritonaeum is interior, in intrathecal, intra-arterial, the ventricles of the brain, In intracerebral, bone, intradermal or intrapulmonary.In some embodiments, it is intravenous for being given and given approach by injection (IV), intramuscular (IM) or subcutaneous (SC).In one embodiment, it is intramuscular injection that parenteral, which is given,.

The total duration at interval and treatment or immunization protocol between determining quantity that low dosage volume gives, giving Within the ability of technical staff.These features can be for example depending on antigen used, desired humoral immunity level, subject And/or disease or illness.

In some embodiments, the low dosage volume of composition give be given during entire immunization protocol by Try people 1,2,3,4,5,6,7,8,9,10 or more time.The composition of low dosage volume is only given in a more specific embodiment, Give the experimenter twice.For example, in one embodiment, can be used as give (priming for the first time for the first time Administration it), and for the second time can be used as reinforcement to give.As used herein, it " gives for the first time " and is directed to subject first It is secondary to give composition, have the effect of antigen presentation to immune system.In contrast, " reinforcement is given " is directed to subject It is then introduced back into same antigen for the second time or arbitrarily.Although first and in reinforcing giving antigen be it is identical, In some embodiments, other components of composition be can be different.

Give being spaced in the ability of technical staff between the composition of low dosage volume.In one embodiment, Interval close enough should to avoid the significant decline of induction immune response.In some embodiments, in composition For the first time give after, when then giving every time about 12 hours after immediately previous (immediately preceding) gives, about 1 day, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 In week or longer time.In a more specific embodiment, every time then low dosage volume give be immediately it is previous give after In about 4 weeks, about 8 weeks or about 12 weeks, and in further embodiment, be immediately it is previous give after about 8 weeks (56 days) when.

In one embodiment, method disclosed herein include be given only single low dosage volume give for the first time with it is single Low dosage volume reinforces the composition given.In one embodiment, it is single for the first time give after about 56 days, mentioned to the experimenter It is given for single reinforcement.

In another embodiment, method disclosed herein includes this paper public affairs for being given only single low dosage volume and giving The composition opened.

The duration for giving composition from giving for the first time to last time in the ability of technical staff, but not It answers too long to encounter ill effect or event.In some embodiments, from first time to the duration given for the last time It is about 0 day (single to give embodiment), about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 The moon, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year or longer.In an embodiment party In formula, the duration is about 8 weeks (56 days).

In one embodiment, method disclosed herein can further comprise the group in low dosage volume as described herein After closing the giving at least once of object, the antigen with post dose is given to the experimenter --- it prepares and is not including hydrophobic carrier or do not wrapping In water-based composition containing hydrophobic carrier and amphipathic compound.In the embodiment of these methods, low dosage volume composition It will be " storage (depot-forming) vaccine " and water-based composition will be " non-stockpiling vaccines ".Illustrative methods and composition It is disclosed in the international patent application no PCT/CA2016/050487 submitted such as on April 27th, 2016.

From " stockpiling vaccines ", mean after being given to the experimenter, vaccine and its component (such as antigen, adjuvant etc.) are in epidemic disease Localization a period of time, the entire body without being rapidly dispersed into subject are kept at seedling injection site.This is referred to herein as " storage effect ", whereby within the extended period from a large amount of released antigens in injection site or antigen and one or more other epidemic diseases Seedling component will not occur.It is desirable that antigen or antigen and one or more other vaccine components are maintained at the period at injection site Certainly in how realizing storage effect.As used herein, term " stockpiling vaccines " is broadly it is meant that most of (substantial Proportion antigen or antigen and one or more other vaccine components) is kept the longer time at injection site If section --- compared to giving antigen and other components in the form of non-stockpiling vaccines.

From " non-stockpiling vaccines ", mean after being given to the experimenter, vaccine and its component (such as antigen, adjuvant etc.) quilt It is dispersed in subject's body rapidly, and " storage effect " can not achieve or not lasting.For non-stockpiling vaccines, antigen or antigen and One or more other vaccine components are quickly removed from injection site significantly than the respective component of stockpiling vaccines.Therefore, non-storage It deposits vaccine and the immune system for contacting antigen in the past is exposed to antigen and other vaccine components again rapidly, then from injection part It dissipates position.In one embodiment, as about 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 are small When, non-stockpiling vaccines greater than 50%, 60%, 70%, 80%, 90% or 100% antigen or antigen and it is one or more its Its vaccine component is removed from injection site.

In one embodiment, method disclosed herein be included in the experimenter be exposed to virus, bacterium, protozoan or Before or after toxin or before and after, the composition of low dosage volume is given to the experimenter.Composition is available wherein To make in the embodiment of preventative vaccine, method is included in the experimenter and is exposed to before virus, bacterium, protozoan or toxin, The composition of low dosage volume at least once is given to the experimenter.

The experimenter can have any gender and any age.In one embodiment, the experimenter be baby, child, Teenager, adult or aged subjects.In some embodiments, the experimenter is the year of 0-2 years old, 50-64 years old or over-65s Age.

The composition that can be used for method disclosed herein is described below.In one embodiment, composition is not Aqueous or generally water-free oil-based composition.

Composition

Based on context requirement, as used herein, term " vaccine ", " vaccine composition " or " composition " is interchangeably It uses.

Composition for method disclosed herein includes the antigen containing B cell epitope, amphipathic compound and hydrophobic load Body.Each of these components is herein by more detail and in a manner of may include exemplary additional component in the composition It is individually described, without limiting.In order to distinguish with other compositions, these compositions are claimed in various situations herein For (such as in instances) " oil base " composition.

In one embodiment, as described herein, composition is not aqueous or generally not aqueous.

Composition is used for method disclosed herein with low dosage volume.In this context, term " therapeutically effective amount " is Refer to the amount (such as Microgram) of the single component in the composition of low dosage volume.Express " therapeutically effective amount " and dose volume without It closes.As used herein, " therapeutically effective amount " means effective stimulus, induction, maintenance, reinforcement or enhancing antibody mediated immunity in subject The amount of the effective component (such as antigen) of response.In some embodiments, the therapeutically effective amount of vaccine is that can have in treatment Cause the amount of clinical response in the subject of body disease or illness.Ability of the determination of therapeutically effective amount in those skilled in the art It is interior, in particular according to disclosure provided herein.Therapeutically effective amount can (patient's condition of such as subject, body depending on various factors Weight, gender and age) and change.

I) antigen

The compositions disclosed herein includes one or more antigens.At least one antigen includes at least one B cell table Position.May include or may not include other antigens of B cell epitope may include in the composition.

At least one of composition antigen includes B cell epitope.It may include more than one B according to the length of antigen Cell epitope.Such as and without limitation, antigen may include one, two, three, four or five B cell epitope.

B cell epitope or epitope can have any chemical property, including but not limited to peptide, carbohydrate, lipid, glycopeptide And glycolipid.In a specific embodiment, epitope is the peptide of one or more antigens derivative described herein or known in the art. Epitope can be identical as naturally occurring epitope, or can be the modified forms of naturally occurring epitope.

B cell epitope is identified by B cell and by antibody.The length of B cell peptide epitopes is generally at least 5 amino acid, more Generally at least 6 amino acid are still more frequently at least 7 or 8 amino acid, and can be continuous (" linear ") or Discontinuous (" conformation ")；The latter is the non-adjacent part object for for example making primary amino acid sequences by protein folding It manages close and formation.Comformational epitope can be identified as linear epitope, such as when they are so synthetically prepared.Generally, linear B is thin The length of born of the same parents' epitope changes between 5-20 amino acid.B cell epitope is also possible to carbohydrate epitope (carbohydrate epitopes)。

Confirm that the experimental method of B cell epitope is known in the art.Generally, when confirming B cell epitope, there are two Importance needs to consider.First aspect is the primary amino acid sequences of protein.The second aspect is the conformation of protein Structure.

The antigen of composition as described herein can be made of or the B cell epitope for capableing of induction body fluid immune response comprising energy The B cell epitope of enough induction body fluid immune responses.

In some embodiments, the antigen in composition as described herein can by relevant to infectious diseases a kind of or A variety of B cell epitopes constitute or include one or more B cell epitopes relevant to infectious diseases.For example, antigen can be by spreading out It is born from virus, such as the B cell epitope of such as influenza virus, zika virus or respiratory syncytial virus (RSV) constitutes or comprising being derived from Virus, such as the B cell epitope of such as influenza virus, zika virus or respiratory syncytial virus (RSV).In one embodiment, B Cell epitope can be the epitope of the hemagglutinin glycoprotein derived from H5N1 influenza virus.In another embodiment, B cell Epitope can be the epitope of the extracellular domain (SHe) of the small hydrophobin derived from respiratory syncytial virus (RSV).

In another embodiment, the antigen in composition as described herein can be by being derived from bacterium, such as such as one hundred days The B cell epitope of cough Bao Te bacillus (Bordetella pertussis) or bacillus anthracis (Bacillus anthracis) is constituted Or comprising being derived from bacterium, such as the B cell epitope of such as pertussis Bao Te bacillus or bacillus anthracis.In one embodiment, B Cell epitope can be the epitope of the pertussis toxoid protein generated by pertussis Bao Te bacillus.In another embodiment In, B cell epitope can be the table of anthrax recombinant protective antigen (rPA) or anthrax mutant recombination protective antigens (mrPA) Position.

In another embodiment, the antigen in composition as described herein can be such as derivative by being derived from protozoan It is constituted from the B cell epitope of Plasmodium (genus Plasmodium) or comprising being derived from protozoan, it is former to be such as derived from malaria The B cell epitope of Eimeria.

In further embodiment, composition may include the mixture of B cell epitope as immune for induction body fluid The antigen of response.B cell epitope can be connected to form single molecule (such as a polypeptide) or (such as separate as separation molecule Polypeptide) exist.

Other than the antigen comprising B cell epitope, in some embodiments, composition as described herein may include containing There is the antigen of CTL epitope.In one embodiment, the sequence that the sequence of CTL epitope can completely or partially with B cell epitope Column overlapping.

CTL epitope is the molecule identified by cytotoxic T lymphocyte.CTL epitope is generally present in antigen presenting cell Surface on, it is compound with MHC molecule.As used herein, term " CTL epitope " refers to natural with antigen (including haptens) Substantially the same molecule of CTL epitope (such as peptide).Compared with the natural negative body (counterpart) of CTL epitope, CTL epitope It can be modified, such as pass through one or two amino acid.Unless otherwise stated, the CTL epitope being mentioned above refer to it is unbonded Molecule, which by cellular uptake and can be present on the surface of antigen presenting cell.

CTL epitope generally should be such epitope, and compliance (amenable) is identified in by T cell receptor, so that can send out Raw cell-mediated immune response.For peptide, CTL epitope can interact with I class or II class MHC molecule.By MHC I class molecule The CTL epitope of presentation is usually peptide of the length between 8 and 15 amino acid, and more generally length in 9 and 11 amino Between acid.CTL epitope by MHC II class molecular presentation is usually peptide of the length between 5 and 24 amino acid, and more logical Normal ground length is between 13 and 17 amino acid.If antigen is bigger than these sizes, will be processed by immune system has It is more suitable for the segment of the size with MHC I class or II class interaction of molecules.Therefore, CTL epitope can be bigger than above-mentioned peptide Peptide part.

Many CTL epitopes are known.Several technologies for identifying additional CTL epitopes by recognize that.In general, These are related to preparation and potentially provide the molecule of CTL epitope and characterize the immune response for being directed to the molecule.

In one embodiment, the antigen in composition as described herein can be made of CTL epitope or comprising CTL table Position.For example, antigen can be by being derived from virus, such as the CTL epitope of HPV or influenza composition or comprising being derived from virus, such as HPV or stream The CTL epitope of sense.In another embodiment, CTL epitope can be the epitope of tumor correlated albumen matter, such as example, herein One or more survivin peptides or melanoma GAP-associated protein GAP.In further embodiment, composition may include CTL The mixture of epitope.CTL epitope can be connected to form single molecule (such as a polypeptide) or (such as separate as separation molecule Polypeptide) exist.

In some embodiments, B cell and CTL epitope are that disease is related and/or disease specific epitope.These diseases Include, but are not limited to any disease as described herein.For example, and without limitation, disease can be infectious diseases (e.g., example Such as, by human papilloma virus (HPV) infection, respiratory syncytial virus (RSV) (RSV) infection, influenza infection, zika virus Infection, Ebola virus infection, infection due to Bacillus anthracis or malariae infect caused or relative disease)；Cancer (e.g., for example, breast cancer, oophoroma, prostate cancer, spongioblastoma or diffusivity large B cell lymphoid tumor)；Or additive disease Sick (e.g., for example, to ***e habituation).

Composition as described herein includes at least one antigen comprising at least one B cell epitope, and can be identical Or further do not include epitope tag (B cell or CTL) on synantigen, it can connect to form single molecule (such as more than one Peptide) or as separation molecule (such as isolated polypeptide) exist.Exemplary antigens comprising such epitope are below without limitation Description.

As used herein, term " antigen " refers to any substance for the component that may specifically bind immune system or divides Son.At least one of composition herein antigen include B cell epitope and can in the experimenter induction of antibodies be immunized answer It answers.It is said that can induce the antigen of immune response is immunogenicity, and may be additionally referred to as immunogene.Therefore, such as this paper institute With term " antigen " includes immunogene, and unless otherwise specified, otherwise term is interchangeably used.As used herein, Term antigen further includes haptens.As understood in the art, haptens be have it is antigenic (such as can be by immune system Component combination) but do not have immunogenicity small molecule, unless its be attached to provide immunogenicity certain carrier molecule.

The antigen that can be used in compositions disclosed herein includes, such as and without limitation, polypeptide, carbohydrate, Microorganism or part thereof, such as living, attenuation, inactivation or kill bacterium, virus or protozoan, or part thereof.Antigen It can be, for example, pathogenic organism preparation, toxin, allergen, peptide, suitable natural, non-natural, recombination or denaturation Protein or polypeptide or its segment or epitope --- can be induced in subject or booster immunization response.In some implementations In mode, antigen be can be derived from animal (animal antigen) --- as such as people (human antigen) --- antigen or in fact Relevant antigen in matter.

As used herein, term " being derived from " includes: and directly separates from primary source (such as subject) without limitation Or the antigen obtained；Identical as the antigen from primary source or substantially relevant synthesis or the antigen being recombinantly produced；Or The antigen made of the antigen of primary source or its segment.In this context, term " substantially related " means that antigen may It is modified by chemistry, physics or other means (such as sequence modification), but products therefrom still is able to generate for original antigen Or the immune response of disease relevant to original antigen or illness.

As used herein, term " antigen " further includes the polynucleotides that coding is used as the polypeptide of antigen.It is known to be based on nucleic acid Vaccination strategies, wherein the vaccine composition containing polynucleotides is administered to subject.By the antigenicity of polynucleotide encoding Polypeptide is expressed in subject, so that antigenic polypeptide is ultimately present in subject, it just look like that vaccine composition itself has contained There is polypeptide the same.For purposes of this disclosure, the term " antigen " indicated within a context includes that this coding is used as antigen The polynucleotides of polypeptide.

In some embodiments, antigen can be antigen relevant to infectious diseases, cancer or addictive disorders.

Can be used as virus of antigen or part thereof in composition herein includes for example, and being not limited to, respiratory syncystial Precursor virus, human airway syncytial virus, influenza virus (such as H5N1 influenza virus, influenza A virus, influenza B disease Poison, influenza virus C), zika virus, human papilloma virus (HPV), vaccinia virus, vaccinia virus, pseudocowpox virus, blister It is exanthema virus, 1 type of nerpes vinrus hominis, 2 type of nerpes vinrus hominis, cytomegalovirus, human adenovirus A-F type, polyomavirus, thin Small virus, hepatitis A virus, hepatitis type B virus, Hepatitis C Virus, human immunodeficiency virus (HIV), positive reovirus It is category, rotavirus, Ebola virus, parainfluenza virus, measles virus, mumps virus, rubella virus, Pneumovirus, mad Dog disease virus, california antigenic group viruses, japanese encephalitis virus, hantaan virus, lymphocytic choriomeningitis virus, Coronavirus genus, Rhinovirus, poliovirus, norovirus (Norovirus), Flavivirus, is stepped on enterovirus genus Remove from office fever virus, West Nile Virus, flavivirus and varicella.

In one embodiment, compositions disclosed herein includes anti-derived from respiratory syncytial virus (RSV) (RSV) It is former.

RSV is the most common viral reason (Nair et al., 2010) of acute lower respiratory infection in whole world child, and And nearly all children are infected when two years old.It infects and occurs in the entire service life again, there is significant phase in the elderly The disease incidence (Walsh and Falsey, 2004) of pass and serious immunocompromised host (Khanna et al., 2008).In Canada, in children The relevant lower respiratory tract of RSV sick (LRTI) causes to be hospitalized per year over 12,000 in child, up to the 2% of birth cohort (Langley et al., 2003).Although having carried out the research of many decades, have shown that fatefully change is established without intervening Rsv infection natural history, therefore nurse generally it is supportive.Understanding RSV epidemiology, virology, disease Huge progress is had been achieved in terms of immune response in pathogenesis and the mankind, but safely effectively RSV epidemic disease is not yet received Seedling.RSV vaccine has been acknowledged as high Global Priority grade (Nair et al., 2011), and it has been proposed that kinds of experiments scheme with gram Take the obstacle of vaccine development.

Nineteen fifty-seven identification virus after soon start develop RSV vaccine (Chanock and Finberg, 1957；Chanock, Roizman and Myers, 1957).A kind of RSV vaccine of Formalin inactivation (refering in particular to (designated) lot number 100) passes through flesh Injection is administered to preschool child and baby to nurse infection (medically in 1966-1967 winter preventive medicine in meat attended infection).The RSV correlation of the baby receptor of RSV vaccine is hospitalized frequency than compareing higher, and two babies Die of rsv infection.To in the further investigation of these children, rodent models repetition experiment and in vitro study led To (led to) this hypothesis: the vaccine of Formalin inactivation generates the water of insufficient serum neutralizing antibody, low-affinity antibody It is flat, cellullar immunologic response or local immunity are not generated, the immune response for formalin modification albumen is changed The induction (Karron, 2013) of (Moghaddam, 2006) and Th2 immune response.For decades, this vaccine related disease enhances The phenomenon that greatly change the situation of RSV vaccine development.

So far, RSV vaccine candidate object included intranasal administration attenuated live it is biologically-derived, genetic engineering, And the subunit vaccine of subunit vaccine and injectable.Many vaccines are absorbed in stimulation of host to generate and be directed to RSV shell egg The neutralizing antibody of white F and G.The progress of molecular biology already leads to the specific RSV antigenicity group that human immune can be directed to Point or epitope identification (Anderson et al., 2010).Due to the vaccine that do not ratify in the market, for safely, effectively and having There are unsatisfied needs for the exploitation of cost-benefit RSV vaccine and availability.

RSV virion (member that paramyxovirus section belongs to) is made of the single-stranded negative justice RNA with 15,222 nucleotide. Three kinds of cross-film surface proteins of nucleotide coding (F, G and small hydrophobin or SH), two kinds of stromatins (M and M2), three kinds of nucleocapsids Body protein (N, P and L) and two kinds of non-structural proteins (NS1 and NS2).So far, the subunit vaccine packet tested in the mankind The F- glucoprotein vaccine for including purifying, combination F, G and M protein vaccine developed by Sanofi Pasteur；G glycoprotein peptide is conjugated (BBG2Na) vaccine and chimeric RSV FG amalgamation protein vaccine.

In one embodiment, compositions disclosed herein may include for the anti-of any one or more of rsv protein It is former.In a specific embodiment, composition includes the SH albumen of RSV or the antigen of its segment.In other embodiments, it combines Object may include the SH albumen of another paramyxovirus or the antigen of its segment.

SH albumen --- being present in multiple paramyxovirus (Collins et al., 1990) --- is with extracellular domain Or the transmembrane protein of " extracellular " component.People's RSV SH albumen contains 64 amino acid (A subgroup) and 65 amino acid (Asias B Group) and be highly conserved.

People RSV SH (A subgroup):

MENTSITIEFSSKFWPYFTLIHMITTIISLLIIISIMIAILNKLCEYNVFHNKTFELPRARVNT(SEQ ID NO:4)

People RSV SH (B subgroup):

MGNTSITIEFTSKFWPYFTLIHMILTLISLLIIITIMIAILNKLSEHKTFCNKTLEQGQMYQINT(SEQ ID NO:5)

Although the function of SH is unclear for many years, recent evidence show that SH has seemed viral porin (viroporin) effect, and indicate inflammatory corpusculum (inflammasome) activation during rsv infection and lure Membrane permeability of the guide pin to ion or small molecule.In Ghent, Belgium university (University of Ghent), independent studies machine Structure Vlaams Instituutvoor Biotechnologie (VIB；Flanders, Belgium) and Baylor College Medicine The researcher of (Baylor College of Medicine) (Houston TX) work has explored in preclinical study Using SH antigen as potential vaccine candidate object.VIB group has specifically had checked outside 23 extracellular domain amino acids of SH albumen Domain portion, referred to as SHe (see, for example, WO 2012/065997).However, SHe is small peptide, therefore individual SHe's is immune Originality is limited.

In one embodiment, compositions disclosed herein includes such antigen --- and it includes the SH of paramyxovirus The extracellular domain (SHe) or its segment structure of the extracellular domain (SHe) of albumen or its segment or the SH albumen by paramyxovirus At.In one embodiment, SHe is derived from ox RSV.In other embodiments, SHe is derived from RSV plants of A subgroup people or B RSV plants of subgroup people.

A subgroup people RSV SHe (RSV SHe A):

NKLCEYNVFHNKTFELPRARVNT(SEQ ID NO:1)

B subgroup people RSV SHe (RSV SHe B):

NKLSEHKTFCNKTLEQGQMYQINT(SEQ ID NO:2)

Therefore, in some embodiments, compositions disclosed herein includes a kind of antigen, which includes amino acid sequence Column NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1) or its segment are made of it.In some embodiments, originally Composition disclosed in text includes a kind of antigen, which includes amino acid sequence NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) or its segment or be made of it.

In some embodiments, composition includes by amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1) the antigen constituted.

In some embodiments, composition includes by amino acid sequence NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) constitute antigen.

In some embodiments, the amino acid sequence of SHe can be modified by insertion, missing and/or amino acid substitution.? In these and other embodiment, SHe may include be at least 50% with the identity of the SHe of SEQ ID NO:1 or 2, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% Amino acid sequence.For example, can be in BLASTp than Alignment measuring sequence alignment (Altschul et al., 1997).For example, in WO The illustrative embodiments of the variant of SHe sequence are disclosed in 2012/065997 (see, for example, in WO 2012/065997 The sequence of SEQ ID Nos:3-30).

In some embodiments, the antigen comprising SHe may include amino acid sequence NKLSEYNVFHNKTFELPRARVNT It (SEQ ID NO:6) or is made of it.In some embodiments, composition includes by sequence The antigen that NKLSEYNVFHNKTFELPRARVNT (SEQ ID NO:6) is constituted.

It, can be with monomeric form, two when compositions disclosed herein includes the antigen containing SHe or its segment or variant Dimer form or another oligomeric forms, or any combination thereof be present in composition.In one embodiment, include The antigen of SHe A and/or SHe B are monomer (such as single polypeptides (single polypeptide)).In another embodiment party In formula, the antigen comprising SHe A and/or SHe B is dimer (such as two individual polypeptides of dimerization).The hand of dimerization Section is known in the art.Exemplary process is that RSV SHe peptide antigen is dissolved in 10%DMSO/0.5% acetic acid aqueous solution (w/ W) it is heated overnight in mixture and at 37 DEG C.

In one embodiment, compositions disclosed herein contains RSV SHe A or its segment or variant conduct The antigen of monomer.

In one embodiment, compositions disclosed herein contains RSV SHe A or its segment or variant conduct The antigen of dimer.

In one embodiment, compositions disclosed herein contains RSV SHe B or its segment or variant conduct The antigen of monomer.

In one embodiment, compositions disclosed herein contains RSV SHe B or its segment or variant conduct The antigen of dimer.

As for example described in WO 2012/065997, SHe peptide antigen can chemically be connect on gene or with carrier. Be suitable for present peptide antigen carrier illustrative embodiments be it is known in the art, some of them are described in WO 2012/ In 065997.In another embodiment, SHe peptide antigen may be connected to amphipathic compound as described herein or be formed by it Structure.

In another embodiment, compositions disclosed herein includes the antigen derived from influenza virus.Influenza is just The single strand RNA virus of glutinous Viraceae and two kinds big glycoprotein, hemagglutinin (HA) and the mind being often based on virion outside It is characterized through propylhomoserin enzyme (NA).Many HA hypotype (Kawaoka et al. 1990 of Flu-A are identified；Webster et al. 1983).In some embodiments, antigen can be derived from HA or NA glycoprotein.In a specific embodiment, antigen can be weight HA antigen (H5N1, the A/Vietnam/1203/2004 of group；Protein Sciences；USA), as logged in derived from Genbank The sequence found under number AY818135 or its any suitable sequence variants.

In another embodiment, compositions disclosed herein includes the antigen derived from Ebola virus.

In another embodiment, compositions disclosed herein includes the anti-of derived from human papillomavirus (HPV) It is former.Compositions disclosed herein includes cervical carcinoma relevant to HPV or the relevant neck of HPV in a more specific embodiment, Cancer-Related antigen.In some embodiments, antigen is such peptide, and it includes sequence RAHYNIVTF (HPV16E7 (H- 2Db) peptide 49-57；R9F；SEQ ID NO:7).

Can be used as bacterium of antigen in confectionery composition or part thereof includes for example, and being not limited to, anthrax (anthrax bar Bacterium), Brucella, pertussis Bao Te bacillus, Mycotoruloides, chlamydia pneumoniae, chlamydia psittaci, cholera (Cholera), Clostridium botulinum, posadasis spheriforme, Cryptococcus, diphtheria (Diphtheria), Escherichia coli O 157: H7, enterorrhagia Property Escherichia coli, enterotoxigenic E.Coli, haemophilus influenzae, helicobacter pylori, Legionnella, Leptospira, benefit This special Pseudomonas, meningococcus (meningococcus), mycoplasma pneumoniae (Mycoplasma pneumoniae), mycobacteria Category, pertussis (Pertussis), pneumonia (Pneumonia), Salmonella, Shigella, staphylococcus (Staphylococcus), streptococcus pneumonia and yersinia enterocolitica.

In one embodiment, compositions disclosed herein includes the antigen derived from bacillus anthracis.Without limitation, The antigen contained in composition can be for example derived from anthrax recombinant protective antigen (rPA) (List Biological Laboratories, Inc.；Campbell, CA) or anthrax mutant recombination protective antigens (mrPA) (Pfenex, Inc.； San Diego, CA).In some embodiments, antigen can derived from the sequence found under Genbank accession number P13423 or Its any suitable sequence variants.

Can be used as protozoan of antigen in confectionery composition or part thereof include for example, and be not limited to, cause malaria Plasmodium (plasmodium falciparum, malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi (Plasmodium knowlesi))。

In one embodiment, compositions disclosed herein includes the antigen derived from malariae.

Optionally, antigen can be natural or synthetic toxin or allergen.As used herein, " toxin " refers to Appointed by what the cell or biology (such as plant, animal, microorganism etc.) that can cause the work of disease or slight illness (ailment) generated What substance or infectious substance or the recombinantly or synthetically molecule that ill effect can be generated.Toxin can be such as small molecule, Peptide or protein.Toxin include drug substance such as, for example, ***e.Toxin can be neutralized by antibody.In such implementation In mode, antigen can cause the generation for being integrated to or completely cutting off the antibody of the toxin in circulation (such as blood), thus potentially pre- Prevent that it is delivered to other regions (such as brain) of body.

As used herein, " allergen " refers to any substance that can be caused allergic reaction.Allergen can be derived from, unlimited In, cell, cell extract, protein, polypeptide, peptide, polysaccharide, polysaccharide conjugates, polysaccharide peptide and Non-peptide mimics, Yi Jizhi Object, animal, fungi, insect, food, drug, dirt (dust) and mite other molecules, small molecule, lipid, glycolipid and carbon aquation Close object.Allergen includes but is not limited to the air-source allergen of environment；Plant pollen (such as artemisiifolia/pollinosis)；Weeds Powder allergen；Careless pollen allergen；Johnson grass sorghum (Johnson grass)；Set pollen allergen；Rye grass (ryegrass)； Arachnid (spider, arachnid) allergen (such as house dust mite allergen)；Storeroom mite allergen；Japanese cedar (Japanese cedar) pollen/pollinosis；Mould/fungal spore allergen；Animal allergen (such as dog, cavy, hamster, The allergens such as gerbil jird, rat, mouse)；Food allergens (such as crustacean；Nut；Citrus fruit；Flour；Coffee)；Elder brother Worm allergen (such as flea, cockroach)；Poisonous substance: (Hymenoptera, yellow jacket (yellow jacket), honeybee, wasp, hornet (hornet), fiery ant)；Bacterial allergen (such as streptococcal antigens；Parasitic animal and plant allergen, such as Ascaris antigen)；Viral antigen； Drug allergen (such as penicillin)；Hormone (such as insulin)；Enzyme (such as streptokinase)；With can be used as incomplete antigen or The drug or chemicals (such as acid anhydrides and isocyanates) of haptens.

When haptens is in compositions disclosed herein, carrier as described herein or other antigens may be affixed to (such as such as protein) is to form hapten-carrier adduct.Hapten-carrier adduct can cause immune response, and partly Antigen itself will not generally cause response.The non-limiting example of haptens is aniline, laccol (toxin in poison ivy), hydrazine Bend piperazine, fluorescein, biotin, foxalin and dinitrophenol.

In another embodiment, antigen can be with need in the circulating cycle sequestered antigen such as such as amyloid protein The relevant antigen of disease (such as Alzheimer disease).Therefore, in some embodiments, composition packet as disclosed herein The antigen that can be potentially served as that neurodegenerative disease is treated and/or prevented in subject in need thereof is included, wherein nerve Degenerative disease is related to the expression of antigen.

In another embodiment, antigen can be derived from cancer or tumor correlated albumen, such as example, can be known by antibody Other film surface combination cancer antigen.

In one embodiment, cancer can be the cancer as caused by pathogen (such as virus).It is contacted with cancer exploitation (linke to) virus be that technical staff is known and include, but are not limited to human papilloma virus (HPV), John Cunningham virus (JCV), human herpes virus 8, angstrom bar virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus With human T cell leukemia virus -1.Therefore, in one embodiment, compositions disclosed herein may include being derived from and cancer The antigen of the virus of disease exploitation connection.It can include expressing one kind from the benefited exemplary cancers of method disclosed herein and composition Or any pernicious cell of kinds of tumors specific antigen, such as the film surface combination cancer antigen that expression can be identified by antibody Cancer.

Much cancers or the relevant albumen of tumour are known in the art, such as example, and being not limited to, WO2007/041832 With those of described in International Application Serial No. PCT/CA2016/050487.For example, the relevant antigen of cancer can derived from HPV (such as E6, E7, L1 or L2 albumen；Or WO1993/022338, WO2002/070006, WO2006/115413, WO2008/147187, One of HPV antigen disclosed in WO2009/002159 or WO2010/123365 is a variety of) or film surface binding protein Survivin (see, for example, WO2014/153636, WO 2004/067023 and WO 2006/081826) or its variant or piece Section.If these antigens include B cell epitope or if they are used as additional antigen in the composition, they may include In compositions disclosed herein.

In some embodiments, composition includes the antigen of weak immunogene.As used herein, from " weak immunogene " It sees, means in conventional vaccine (such as aqueous vaccine, emulsion etc.), antigen has little or no induction, maintains and/or add The ability of strong immune response.

For example, in one embodiment, weak immunogene antigen cannot be sufficiently induced when being prepared with aqueous vaccine The antigen of immune response.This and same antigen (have same composition, remove with comparable vaccine composition as disclosed herein Prepare in the hydrophobic carrier with amphipathic compound) when preparing on the contrary, thus antigen can sufficiently induce immune response now. In aforementioned context, " sufficiently induction immune response " means that antigen induction of antibodies immune response can reach it in the experimenter Lead to the degree of the detectable antibody titer as described in present example (i.e. under the detection limit of 1/100 dilution).

In one embodiment, weak immunogene antigen is this antigen, is being exposed in a manner of aqueous vaccine After subject, immune response or induction is not induced or not with after being exposed to subject in a manner of composition as described herein Immune response compared to low at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more of effect immune response.

In one embodiment, weak immunogene antigen is this antigen, and when being given with aqueous vaccine, it cannot be right Subject provides measurable treatment benefit；And it is able to achieve when antigen is given with composition as disclosed herein measurable Treatment benefit.In one embodiment, for example, measurable treatment benefit can be in detection limit as described herein With the antibody mediated immunity response of detectable antibody titer under (1/100 dilution).

Without limitation, weak immunogene antigen may include, for example, purify and antigenic synthetic peptide；Autoantigen；Cancer Related antigen；Or neoantigen (neoantigens).

In one embodiment, compositions disclosed herein is included as the antigen of autoantigen.As known in the art , autoantigen is derived from the intracorporal antigen of subject.Immune system is not directed to autoantigen usually under the conditions of normal steady state Reaction.Therefore, the antigen of these types causes difficulty in the exploitation of targeting immunotherapy.

In one embodiment, compositions disclosed herein includes neoantigen.It may include neoantigen in the composition Be, such as and without limitation, the U.S. Provisional Application No. 62/331 on May 4th, 2016 is filed in, those of described in 770.

In one embodiment, the antigen contained in composition may include the mixed of one or more antigens as described herein Object is closed, is optionally fused together as fusion protein, wherein with or without spacer sequence between antigen.

In one embodiment, antigen is the polypeptide derived from any antigen as described herein.In an embodiment In, antigen is the peptide antigen that length is 5 to 50 amino acid.

For example, and without limitation, can be used as the antigen in confectionery composition polypeptide or its segment include derived from Under polypeptide or its segment: extracellular domain, cholera toxoid, the tetanus toxoid, diphtheria of small hydrophobic (SH) albumen of RSV Toxoid, hepatitis B surface antibody, hemagglutinin (such as hemagglutinin of H5N1 recombination), anthrax recombinant protective antigen (List Biological Laboratories, Inc.；Campbell, CA), anthrax mutant recombinate protective antigens (Pfenex, Inc.；San Diego, CA), neuraminidase, influenza M albumen, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1, Der-p-1, Der-f-1, fat differentiation related protein (Adipophilin), AFP, AIM-2, ART-4, BAGE, first tire Albumen, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin D1 Ep-CAM, EphA2, EphA3, ELF-2, FGF-5, G250, gonadotropin-releasing hormone (GRH) (GNRH), HER-2, enteron aisle carboxy-lesterase (iCE), IL13R α 2, MAGE-1, MAGE-2、MAGE-3、MART-1、MART-2、M-CSF、MDM-2、MMP-2、MUC-1、NY-EOS-1、MUM-1、MUM-2、MUM- 3, pertussis toxoid albumen, p53, PBF, PRAME, PSA, PSMA, RAGE-1, RNF43, RU1, RU2AS, SART-1, SART- 2, SART-3, SAGE-1, SCRN 1, SOX2, SOX10, STEAP1, survivin, Telomerase, TGF β RII, TRAG-3, TRP- 1, TRP-2, TERT and WT1.

Term " polypeptide " includes any amino acid chain, no matter length (for example, at least 6,8,10,12,14,16,18 or 20 Amino acid) or posttranslational modification (such as glycosylation or phosphorylation) how, and including for example, native protein, synthesis or again Group polypeptide and peptide, epitope, hybrid molecule, variant, homologue, analog, peptidomimetic, peptide mimics (peptidomimetics) etc.. Therefore, variant or derivative include missing, including truncate and fracture；Insertion and addition, such as conservative substitution, site directed mutation Body and allelic variant；And modification, including having the one or more non-amino acyl groups for being covalently attached to peptide (for example, sugar, rouge Matter etc.) peptidomimetic and posttranslational modification.As used herein, term " conservative amino acid substitution " or " conservative replaces " refer to One amino acid substitution is another amino acid by given position in peptide, wherein replacing can largely not lose correlation function In the case where carry out.When carrying out this variation, the substitution of similar amino acid residue can be in the relative similarities of side chain substituents It is carried out on the basis of (for example, its size, charge, hydrophobicity, hydrophily etc.), and this substitution can be measured by routine test Its influence to peptide function.The specific non-limiting example of conservative replaces includes following instance:

Original Residue	Conservative replaces
		Ala	Ser
Arg	Lys
		Asn	Gln, His
Asp	Glu
		Cys	Ser
Gln	Asn
		Glu	Asp
His	Asn, Gln
		Ile	Leu, Val
Leu	Ile, Val
		Lys	Arg, Gln, Glu
Met	Leu, Ile
		Phe	Met, Leu, Tyr
Ser	Thr
		Thr	Ser
Trp	Tyr
		Val	Ile, Leu

The polypeptide or peptide that there is Substantial identity with antigen sequence can be used.If worked as in optimal comparison (with the vacancy of permission) When, their shared at least about 50% sequence identity, or if the shared function motif limited of sequence, then it is assumed that two sequences Column have Substantial identity.In an alternate embodiment, if the sequence of optimal comparison is shared at least on specified region 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, then it is believed that they are real (that is, there is Substantial identity) identical in matter.Term " identity " refers to the sequence similarity between two peptide molecules.It is same Property can be determined by comparing each position in aligned sequences.Identity degree between amino acid sequence is shared in sequence Position at (for example, specified region on) identical or the function of the quantity of matching amino acid.The sequence compared for identity Optimal comparison can be carried out using various algorithms known in the art, including ClustalW program (can beHttp: // clustalw.genome.ad.ipObtain), the local homology algorithm (1981) of Smith and Waterman, Needleman and The homology alignment algorithm (1970) of Wunsch, the search (1988) of the similarity method of Pearson and Lipman and these calculations (such as Wisconsin Genetics Software Package, Genetics Computer is implemented in the computerization of method GAP, BESTFIT, FASTA and TFASTA in Group, Madison, WI, U.S.A.).Altschul et al. institute can also be used The BLAST algorithm (using disclosed default setting) stated determines sequence identity (1990).For example, National can be passed through Center for Biotechnology Information (passes through internetHttp:// www.ncbi.nlm.nih.gov/ BLAST/b12seq/wblast2.cgi) obtain " BLAST2 Sequences " tool, with following default setting selection " blastp " program: expectation threshold value 10；Word length (font size, word size) 3；Matrix B LOSUM62；Originate gap penalty (gap Costs existence) 11, extend (extension) 1.In another embodiment, those skilled in the art can only pass through Visual inspection easily and suitably compares any given sequence and infers sequence identity and/or homology.

It can be separated from natural source (natural sources) for practicing polypeptide and peptide of the invention, be synthesis, or Recombinate the polypeptide generated.Peptide and protein can express with recombinating in vitro or in vivo.Peptide and polypeptide for practicing of the invention can Utilize any method preparation known in the art and separation.For practice polypeptide and peptide of the invention can also be used it is known in this field Chemical method entirely or partly synthesize.See, for example, Caruthers 1980, Horn 1980, Banga, 1995.For example, Peptide synthesis can execute (see, for example, Roberge 1995, Merrifield 1997) using various solid phase techniques, and can realize It is automatically synthesized, such as utilizes ABI 431A peptide synthesizer (Perkin Elmer) according to the specification that manufacturer provides.

In some embodiments, antigen can be the antigen of purifying, for example, about 25% to about 50% pure, about 50% to about 75% is pure, about 75% to about 85% pure, about 85% to about 90% pure, about 90% to about 95% pure, about 95% to about 98% pure, about 98% to about 99% is pure or pure greater than 99%.

As described above, term " antigen " also includes the polynucleotides that coding is used as the polypeptide of antigen.As used herein, term " polynucleotides " include any length (such as 9,12,18,24,30,60,150,300,600,1500 or more nucleotide) or The nucleotide chain of chain number (such as single-stranded or double-stranded).Polynucleotides can be DNA (such as genomic DNA or cDNA) or RNA (such as mRNA) or combinations thereof.They can be natural or synthetic (such as chemically synthesizing).It is expected that multicore glycosides Acid may include the modification to one of nucleotide chain or a variety of nitrogenous bases, pentose or phosphate group.These modifications are in ability It is well known in domain, and can be used for for example improving the purpose of the stability of polynucleotides.

Polynucleotides can deliver in a variety of manners.In some embodiments, naked polynucleotides (naked Polynucleotide) can be used --- in linear form, or it is inserted into plasmid (such as expression plasmid).In other embodiment In, carrier living, such as virus or bacteria carrier can be used.

The one or more adjusting sequences for helping for DNA to be transcribed into RNA and/or RNA being translated into polypeptide may be present.? Under some cases, such as in the case where polynucleotides are mRNA (mRNA) molecule, the adjusting sequence for being related to transcription is not needed It arranges (such as promoter), and protein expression can realize (effected) in the case where no promoter.Technical staff can It according to circumstances needs to adjust sequence comprising suitable.

In some embodiments, polynucleotides are present in expression cassette, and wherein it is operably connected to adjusting sequence, The adjusting sequence will allow polynucleotides to be expressed in the subject for giving composition as disclosed herein to it.Expression cassette Selection depend on the subject of composition and the polypeptide of expression are given to it needed for feature.

Generally, expression cassette includes promoter, functional in subject and can be composing type or induction type；Ribose Body binding site；Initiation codon (ATG) (if necessary)；The polynucleotides of encoding target polypeptide；Terminator codon；With it is optional Ground 3' terminal region (translation and/or transcription terminator).It may include appended sequence, such as the region of encoded signal peptide.Encoding target The polynucleotides of polypeptide can with it is any homologous or heterologous in other adjusting sequences in expression cassette.With target polypeptides (as believed Number peptide-coding region domain) sequence that is expressed together be normally at encode protein to be expressed polynucleotides be nearby placed in it is suitable When reading frame in.(such as believed by encoding the polynucleotides of protein to be expressed individually or with any other sequence to be expressed Number peptide) open reading frame that constitutes together is placed under the control of promoter, so that transcription and translation, which is betided, gives group to it In the subject for closing object.

The amount (amount, quantity) for the antigen that single low dosage volume for composition as described herein is given It can be changed according to the type of antigen and the feature (such as size, weight, age, gender etc.) of subject.Those skilled in the art Member will determine the effective quantity of the antigen used in a particular application without excessive experiment.As made herein Term " effective quantity " means the amount that desired result is effectively realized within the necessary period.

In one embodiment, it is given every time to the low dosage volume of the experimenter, composition can include about 5-50 microgram Between antigen.It in some embodiments, is given every time to the low dosage volume of the experimenter, composition includes about 5 μ g, about The antigen of 10 μ g, about 15 μ g, about 20 μ g, about 25 μ g, about 30 μ g, about 35 μ g, about 40 μ g, about 45 μ g or about 50 μ g.In a reality It applies in mode, is given every time to the low dosage volume of the experimenter, composition includes the antigen of about 10 μ g.In another embodiment In, it is given every time to the low dosage volume of the experimenter, composition includes the antigen of about 25 μ g.

In some embodiments, antigen itself is sufficiently hydrophobic, so that antigen can mix in hydrophobic carrier.? In some embodiments, antigen is by sufficiently hydrophobic preparation (such as by amphipathic compound), so that antigen is in hydrophobic carrier It can mix.

Ii) amphipathic compound

" amphipathic compound " is the compound with hydrophilic and hydrophobic (lipophilic) part or feature.Term " amphipathic compound " It can be used interchangeably with " amphiphile " or " amphiphilic ".In some embodiments, suitable amphipathic compound may also include cream Those of agent, as described below.The illustrative embodiments for the emulsifier for including by term " amphipathic compound " herein Including being not limited to, Spheron MD 30/70 (polysorbates) (such as sorbitan monooleate), mannide oleate (Arlacel^TMA), lecithin, Tween^TM80 and Spans^TM20,80,83 and 85.Amphipathic compound, which can promote, not to be had The vaccine component with hydrophilic compatibility is incorporated into hydrophobic carrier (such as oil) in the case where water.Vaccine component may include, no It is limited to, antigen and/or adjuvant and/or can promote the other ingredients for generating immune response.

Without limitation, the hydrophobic part of amphipathic compound is usually big hydrocarbon part, such as CH₃(CH₂)_nThe long-chain of form, Wherein n > 4.The hydrophilic segment of amphipathic compound is usually charged group or polarity without electric group.Charged group includes anion And cation group.The example of anionic charged groups includes following (wherein the hydrophobic part of molecule is indicated by " R "): carboxylic acid Root: RCO₂ ^-；Sulfate radical: RSO₄ ^-；Sulfonate radical: RSO₃ ^-；With phosphate radical (the electrification degree of functionality (functionality) in phosphatide). Cationic-charged group includes such as amine: RNH₃ ⁺(hydrophobic part that " R " represents molecule again).Uncharged polar group includes example Such as with the alcohol of big R group, such as diacylglycerol (DAG).Amphipathic compound can have several hydrophobic parts, several hydrophilic segments, Or it is both several.Protein and certain block copolymers are examples.Steroids, cholesterol, fatty acid, bile acid and saponin(e It is amphiphile.

There are many workable amphipathic compounds, and vaccine composition disclosed herein may include the two of single type The mixture of close compound or different types of amphipathic compound.

In one embodiment, amphipathic compound is lipid or lipid mixture.Although the rouge of any amphiphilic can be used Matter, but specially suitable lipid may include having containing at least four carbon and general length is at least one of about 4 to 28 carbon The lipid of fatty acid chain.Fatty acid chain can contain any amount of saturation and/or unsaturated bond.Lipid can be natural lipid or Synthesize lipid.The non-limiting example of the lipid of amphiphilic may include phosphatide, sphingolipid, sphingomyelins, cerebroside (cerobrocides), Gangliosides, ether rouge, sterol, cuorin, cation lipid and with poly(ethylene glycol) and other polymer-modified lipids.It closes It may include at lipid, be not limited to, following fatty acid composition: lauroyl, cardamom acyl group, palmityl, stearyl, peanut four Enoyl- (arachidoyl), oleoyl, sub-oleoyl (linoleoyl), mustard acyl (erucoyl) or these fatty acid Combination.

In one embodiment, amphipathic compound is the mixture of phosphatide or phosphatide." phosphatide " broadly defined is The member of one group of lipid compounds of (yield) is generated on hydrolysis phosphoric acid, alcohol, fatty acid and nitrogenous base.

Workable phosphatide include for example, and be not limited to, have at least one head base (head group) selected from the following Phosphatide: phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine (such as DOPC；1,2- dioleoyl-sn- glycerol Base -3- phosphocholine) and lipositol (phosphoinositol).In some embodiments, DOPC and no esterification can be used Cholesterol mixture.In other embodiments, the mixed of the cholesterol of Lipoid S100 lecithin and no esterification can be used Close object.When using no esterification cholesterol when, cholesterol can be equivalent to phosphatide weight about 10% amount use (such as with The S100 lecithin (lecitin) of the DOPC: cholesterol ratio or 10:1w/w of 10:1w/w: cholesterol ratio).Cholesterol is used for Stablize the formation of phospholipid vesicles (phospholipid vesicles).If using the compound other than cholesterol, ability Field technique personnel can easily determine required amount.

Another common phosphatide is sphingomyelins.Sphingomyelins includes sphingol, a kind of amino alcohol with long aliphatic unsaturated hydrocarbon. Fatty acyl side chain is connected by the amino of amido bond and sphingol to form ceramide.The hydroxyl of sphingol is esterified into gallbladder Alkali phosphoric acid.Similar phosphoglyceride, sphingomyelins is amphiphilic.

The lecithin that can also be used is the natural mixture for typically being derived from the phosphatide of egg or wool.

In all these and other phosphatide practices for use in the present invention.Phosphatide is purchased from such as Avanti lipids (Alabastar, AL, USA) and lipoid LLC (Newark, NJ, USA).

In one embodiment, amphipathic compound can be substantially homogeneously dispersed in hydrophobic carrier, thus amphiphilic The individualism for closing object is enough to promote for the vaccine component (such as antigen) with hydrophilic compatibility to be incorporated into hydrophobic carrier.

In another embodiment, amphipathic compound can be closely related with antigen, so that antigen can be mixed in hydrophobic carrier In.From " closely related ", mean amphipathic compound and antigen so close to (proximity), so that antigen is being dredged with it It is that the form that can be mixed exists in water carrier.The closely related physics phase that may relate to or may not be related between antigen and amphiphile Interaction.Generally, the hydrophilic segment of amphipathic compound is oriented towards the hydrophilic segment on antigen.Amphipathic compound can be protected generally It holds and is separated from each other or they can form various types of structure, assembly (assemblies) or array.

The illustrative embodiments of the type of structure, assembly or array that amphipathic compound can be formed include being not limited to: single Synusia, double-layer tablets, multilayer tablet, single layer blister (vesicular) structure (such as micella (micelles)), the double-deck balloon-shaped structure (such as single-layer or multi-layer vesicle (unilamellar or multilamellar vesicles)) or its various combination.From " single layer " is seen, means that amphipathic compound does not form bilayer, but is kept as one layer, and wherein hydrophobic part is oriented in one Side and hydrophilic segment is oriented in opposite side.From " bilayer ", mean that amphipathic compound forms double-layer tablets (two-layered Sheet), generally wherein every layer of hydrophobic part is inwardly directed towards double-deck center, and hydrophilic segment is outwardly directed.However, Opposite configuration is also possible.Term " multilayer " mean include single and double layer structure any combination.The form of use can depend on In used specific antigen, specific amphipathic compound and/or specific hydrophobic carrier.

In one embodiment, structure, assembly or the array formed by amphipathic compound can be wrapped partially or even wholly Enclose antigen.As an example, amphipathic compound can form closing balloon-shaped structure around antigen.As another example, in advance The structure (such as precursor (presomes)) being initially formed can be by disassembled/reassembled with encirclement antigen and/or the antigen that swallows up.

In one embodiment, balloon-shaped structure is single layer balloon-shaped structure.The example of this structure is micella.Aqueous solution In typical micelle forma-tion aggregate, wherein hydrophilic segment is contacted with the aqueous solution of surrounding, completely cut off micella center in it is hydrophobic Part.In contrast, in hydrophobic carrier, inverse/reverse micelle is formed, wherein hydrophobic part is contacted with the aqueous solution of surrounding, every Hydrophilic segment in exhausted micella center.Spherical reverse micelles can wrap up the antigen with hydrophilic compatibility in its core.

In one embodiment, balloon-shaped structure is micella or inverse/reverse micelle.Without limitation, micella or inverse/reverse micelle Magnitude range be 2nm (20A) to 20nm (200A) (diameter).In a specific embodiment, the size of micella or inverse/reverse micelle It is about 10nm (diameter).

In another embodiment, balloon-shaped structure is the double-deck balloon-shaped structure, such as example, liposome.Liposome is complete The closed bilayer lipid membrane in ground, it includes (entrapped) aqueous volumes of embedding.Liposome, which can be monolayer vesicle, (to be had Single duplicature) or multi-layer vesicles (being characterized in that multimembrane bilayer)；Each bilayer may or may not by aqueous layer with it is next A separation.The general discussion of liposome can be found in Gregoriadis 1990 and Frezard 1999.Liposome is actually It can be adsorbed onto any kind of cell, then discharge the agent (such as antigen) being incorporated to.Optionally, liposome can melt with target cell It closes, thus the content injection (empty) of liposome is into target cell.Optionally, liposome can be by with phagocytic cell Institute's endocytosis.

Liposome has been used for preparing composition, and the composition includes anti-to encapsulate (encapsulate) as vesicle The emulsifier of former hydrophobic carrier and stabilization formulations is (see, for example, WO2002/038175, WO2007/041832, WO2009/ 039628, WO2009/146523 and WO2013/049941).Hydrophilic antigen is generally embedded in aqueous interior, and hydrophobic anti- In the pluggable double-layer of lipoid of original or it is dispersed in oily phase.In another embodiment, previously fabricated liposome can be used for this In vaccine composition disclosed in text.

In one embodiment, balloon-shaped structure is precursor.As used herein, " precursor " refer to magnitude range be≤ The nano particle that 110nm and polydispersity index (pdi) are < 0.1.Precursor may include for example such liposome, mainly its The middle spherical unilamellar liposome of the double-deck formation, and spherical multilamellar liposome, oval unilamellar liposome, oval multilamellar liposome With the mixture of oval multivesicular liposomes.

Double-deck and multilayer balloon-shaped structure other embodiment includes being not limited to: niosomes (niosomes), carrier (transfersomes), virion, multi-layer vesicles (multilamellar vesicles, MLV), few layer vesica (oligolamellar vesicles, OLV), monolayer vesicle (UV), small monolayer vesicle (SUV), medium-sized monolayer vesicle (MUV), Big monolayer vesicle (LUV), huge monolayer vesicle (GUV), more vesicles steep (MVV), single layer or widow made of reverse phase evaporation Layer vesica (REV), the multi-layer vesicles made of reverse phase evaporation (MLV-REV), stable multilayer (plurilamellar) capsule Bubble (SPLV), freezing and defrosting MLV (FATMLV), by pressing method prepare vesica (VET), pass through French press (French press) preparation vesica (FPV), by melting preparation vesica (FUV), dehydrated-rehydrated vesicles (DRV) and Foam (bubblesomes) (BSV).Technical staff will be recognized that the technology for preparing these balloon-shaped structures is well known in the art (see, for example, Kreuter 1994).

In some embodiments, amphipathic compound can form structure described herein in the single formulation of composition Any combination.For example, composition may include a variety of different structures, assembly or array.In one embodiment, composition can wrap Containing similar liposome bilayer, spherical unilamellar liposome, spherical multilamellar liposome, oval unilamellar liposome and the more bubbles of ellipse The structure of liposome.

Iii) hydrophobic carrier

Compositions disclosed herein includes hydrophobic carrier, preferred liquid hydrophobic substance.

Hydrophobic carrier can be the mixture of substantially pure hydrophobic substance or hydrophobic substance.It can be used for as described herein group Closing the hydrophobic substance in object is acceptable hydrophobic substance pharmaceutically and/or in immunology.Carrier is usually liquid, but big Be not at a temperature of gas liquid certain hydrophobic substances can (such as by heating) be liquefied, and be also possible to useful.

The mixture of oil or oil is the specially suitable carrier for compositions disclosed herein.Oil should be pharmaceutically And/or it is acceptable in immunology.Suitable oil includes, for example, mineral oil (especially lightweight or low-viscosity mineral oil, such as6VR), vegetable oil (such as soybean oil), macadamia nut oil (such as peanut oil), or mixtures thereof.Therefore, in a reality It applies in mode, hydrophobic carrier is hydrophobic substance, such as vegetable oil, macadamia nut oil or mineral oil.Also can be used animal tallow and it is artificial dredge Water polymeric material is especially liquid at atmospheric temperature or those of can relatively easily be liquefied.

In some embodiments, hydrophobic carrier can be or comprising incomplete Freund's adjuvant (Incomplete Freund's Adjuvant, IFA), a kind of mineral oil basic mode type hydrophobic carrier.In another embodiment, hydrophobic carrier can To be or mineral oil solution comprising mannide oleate, such as can be used asISA 51 (SEPPIC, France) That commercially available.Although these carriers are commonly used for preparing water-in-oil emulsion, the disclosure passes through the feelings in not a large amount of water The preparation of the type is avoided under condition using amphipathic compound suspending components, as described herein.

Immunovaccine Inc. has been developed referred to asAnd DepoVax^TM(DPX) vaccine delivery Platform is (see, for example, U.S. Patent number 6,793,923 and 7,824,686；WO2002/038175；WO2007/041832； WO2009/039628；WO2009/043165 and WO2009/146523).DPX is oily packet rouge (lipid-in-oil) preparation, It can be prepared with the mixture of any antigen or antigen.Different from the vaccine based on water-in-oil emulsion --- it contains dependent on oil embedding There is the water droplet of antigen and adjuvant, is based on DepoVax^TMPreparation dependent on lipid to promote antigen and adjuvant being incorporated directly into oil In, without emulsification.The advantages of program includes: dissolution of (1) enhancing hydrophilic antigen/adjuvant in oily diluent Property, in addition to this hydrophilic antigen/the adjuvant has maximum solubility usually in water-based diluent, and (2) are given in vaccine Interminable emulsification procedure is eliminated before giving.

In some embodiments, vaccine composition disclosed herein may include that the delivering of Immunovaccine Inc. is flat Platform DepoVax^TM。

In one embodiment, composition includes: (i) includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT The peptide of (SEQ ID NO:1)；(ii) comprising the lipid mixture of dioleyl phosphatidyl choline (DOPC) and cholesterol；(iii) Short synthesis lipopeptid, is PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)；And (iv)ISA 51 VG。

Iv) other components

Compositions disclosed herein can further include one or more annexing ingredients known in the art (see, for example, Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985；And The United States Pharmacopoeia:The National Formulary (USP 24NF19), It is published in 1999).

In some embodiments, composition can additionally comprise adjuvant, emulsifier, T auxiliary epitope (t helper cell epitope, T-helper epitope) and/or excipient.

Adjuvant

In some embodiments, compositions disclosed herein may include one or more adjuvants.

A large amount of adjuvant has been described and has been known to the skilled in the art.Exemplary Adjuvants include being not limited to, bright Alum, other compounds of aluminium, BCG vaccine (Bacillus of Calmette and Guerin, BCG), TiterMax^TM、 Ribi^TM, Freund's complete adjuvant (FCA), the oligodeoxynucleotide (CpG ODN) containing CpG, lipid A mimic or its analog, rouge Peptide and polyI:C polynucleotides.

In some embodiments, composition may include lipopeptid as adjuvant.Exemplary lipopeptid includes being not limited to, PAM₃Cys-SKKK (SEQ ID NO:3) (EMC Microcollections, Germany) or its variant, homologue and similar Object.Lipopeptid (such as the PAM of Pam2 family₂Cys-SKKK；SEQ ID NO:3) have been displayed be Pam3 family lipopeptid effectively replace For object.

In some embodiments, composition may include CpG ODN as adjuvant.Exemplary CpG ODN is 5'- TCCATGACGTTCCTGACGTT-3'(SEQ ID NO:8).Technical staff can be on the basis of target species and effect easily Select other CpG ODNs appropriate.

In some embodiments, composition may include PolyI:C polynucleotides as adjuvant.

PolyI:C polynucleotides be containing inosinicacid residue (I) and cytidine monophosphate residue (C) polynucleotide molecule (RNA or The combination of DNA or DNA and RNA), and the generation of its inflammation inducing cell factor (such as interferon).In some embodiments, PolyI:C polynucleotides are double-strands.In such embodiment, they the nucleotide containing cytimidine generally by being made of completely A chain and be made of completely the chain that the nucleotide containing inosine is constituted, although other configurations are possible.For example, every Chain can contain nucleotide of the sum containing cytimidine containing inosine.In some cases, any or two chains can be in addition containing one kind Or a variety of non-cytimidines or non-inosine nucleotide.

In another embodiment, PolyI:C polynucleotides can be containing inosinicacid residue (I) and cytidine monophosphate residue (C) single chain molecule.As an example, and without limitation, single-stranded polyI:C can be the sequence of repetition dIdC.Having In body embodiment, the sequence of single-stranded polyI:C can be (IC)₁₃26 oligomeric sequences, i.e., ICICICICICICICICICICICICIC(SEQ ID NO:9).As the skilled person will understand that, due to their property (example Such as complementarity), it is contemplated that these single chain molecules for repeating dIdC will naturally occur homodimer, therefore they are conceptually similar to PolyI/polyC dimer.

It is reported that polyI:C can be divided with every 16 residues, and (Bobst is not influenced on its interferon activation potentiality 1981).In addition, introducing the interferon inducer of the polyI:C molecule of Uridine residues mispairing by every 12 repetitions cytidine monophosphate residue It leads potentiality (Hendrix 1993) and shows that the minimum double-strand polyI:C molecule of 12 residues is enough that interferon is promoted to generate.It is other It has also been shown that corresponding to the region of as low as 6-12 residue of the 0.5-1 spiral corner (helical turn) of double-stranded polynucleotide Induction Process (Greene 1978) can be triggered.If be synthetically prepared, PolyI:C polynucleotides length be typically about 20 or More residues (22,24,26,28 or 30 residues of normal length).If semi-synthetic preparation (such as using enzyme), chain Length can be 500,1000 or more residues.

PolyI:C serves as virus genomic analogies and is particularly useful for adjusting vivo immuning system.For example, having reported Road, when through intravenously or intramuscularly interior injection systemic delivery in vivo, synthesis poly I:poly C homopolymer passes through non-specific Property inducing interferon γ enhance congenital immunity (Krown 1985, Zhu 2007).Polyinosine for many years has been described With several variants (de Clercq 1978, Bobst 1981, de Clercq 1975, Guschlbauer of cytidine acid polymer 1977, Fukui 1977, Johnston 1975, U.S. Patent number 3,906,092, Kamath 2008, Ichinohe 2007), Some of them include the residue using covalent modification, using ribose and deoxyribose inosine and cytidine residue, using containing inosine The polymer of mispairing is created with the homopolymer and alternate copolymer of cytidine monophosphate residue, and the specific residue of introducing.

It has been reported that using the double-stranded polynucleotide containing inosine and cytidine monophosphate for treating a variety of virus diseases (Kende 1987, Poast 2002, U.S. Patent number 6,468,558, Sarma 1969, Stephen 1977, Levy 1978), cancer (Durie 1985, Salazar 1996, Theriault 1986, Nakamura 1982, Talmadge 1985, Droller 1987), such as other senses of the autoimmunity disease of multiple sclerosis (Bever 1986), and such as malaria Infectious diseases (Awasthi 1997, Puri 1996).By the way that molecule and positively charged polylysine and carboxymethyl cellulose are answered Close, effective protection polynucleotides in vivo from nuclease degradation (Stephen 1977, Levy 1985) or pass through by PolyI:C and positively charged synthetic peptide are compound (Schellack 2006), have further enhanced polyI:C in some cases The effect of molecule.

Other than it is used as the non-specific enhancer of congenital immunity, polyI:C is also acted as in vaccine composition Adjuvant.The enhancing of congenital immunity can lead to the antigentic specificity adaptive immunity of enhancing --- and it may be by being at least partly related to NK Cell, macrophage and/or dendritic cells mechanism (Chirigos 1985, Salem 2006, Alexopoulou 2001, Trumpfheller 2008).In this context, infectious diseases is controlled certainly using the evidence source of polyI:C molecule (Houston 1976, Stephen 1977, Ichinohe 2007, Sloat 2008, Agger 2006, Padalko 2004) With prevented by various vaccine forms or treating cancer (Zhu 2007, Cui 2006, Salem 2005, Fujimura 2006, Llopiz 2008) various vaccine researches.These researchs have shown that polyI:C enhances humoral response (humoral Responses), as from it will become apparent from the antibody response enhanced for specific infectivity disease antigen.PolyI:C is also Antigen-specific cellular response (cellular responses) synergist (Zhu 2007, Zaks 2006, Cui 2006, Riedl 2008).The secondary effects of polyI:C molecule are considered at least partially through them and toll sample receptor (TLR) (such as TLR3, TLR4, TLR7, TLR8 and TLR9) interaction inducing interferon-γ and occur (Alexopoulou 2001, Trumpfheller 2008, Schellack 2006, Riedl 2008), wherein TLR3 and major part polyI:C molecule are special It is related.Evidence be also shown that at least partially by with the receptor other than TLRs --- such as inducible protein matter I (RIG-I)/melanocyte The RNA helicase retinoic acid of tumor differentiation associated gene 5 (MDA5) --- interaction, polyI:C molecule can play its effect (Alexopoulou 2001, Yoneyama 2004, Gowen 2007, Dong 2008).The mechanism of action of polyI:C molecule is still Need to be understood completely.

Therefore, as used herein, " polyI:C ", " PolyI:C polynucleotides " or " PolyI:C polynucleotides adjuvant " is Double or single stranded polynucleotide molecule (combination of RNA or DNA or DNA and RNA), wherein every chain contains the adjacent flesh of at least six Glycosides or cytidine monophosphate residue, or 6 neighbours selected from inosinicacid and cytidine monophosphate (such as IICIIC or ICICIC) in any order The residue connect, and it can induce in mammalian subject or enhance at least one inflammatory cytokine (such as interferon) Generation.PolyI:C polynucleotides will generally have about 8,10,12,14,16,18,20,22,24,25,26,28,30,35, 40,45,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,500,1000 or more residue Length.Preferred PolyI:C polynucleotides can have about 6,8,10,12,14,16,18,20,22,24,26,28 or 30 The maximum of the minimum length of nucleotide and about 1000,500,300,200,100,90,80,70,60,50,45 or 40 nucleotide Length.

Every chain of double-strand PolyI:C polynucleotides can be the homopolymer or every chain of inosine or cytidine monophosphate residue It can be the heteropolymer containing both inosine and cytidine monophosphate residue.In any case, polymer can be by one or more non-fleshes Glycosides or non-cytidine monophosphate residue (such as uridine) interrupt, and condition is the presence of at least one 6 I, 6 C or 6 I/C as described above The neighboring region of residue.Generally, every chain of PolyI:C polynucleotides will be residual containing not more than 1 non-every 6 I/C of I/C residue Base, it is further preferred that not more than 1 non-every 8,10,12,14,16,18,20,22,24,26,28 or 30 I/C residue of I/C residue.

As it is known in the art, inosinicacid or cytidine monophosphate (or other) residue in PolyI:C polynucleotides can be derivatized Or modification, condition are the abilities for retaining PolyI:C polynucleotides and inflammatory cytokine (such as interferon) being promoted to generate.Derivative or The non-limiting example of modification is included such as azido modification, fluoro modification or is replaced using thioesters (or similar) key natural Phosphodiester bond is with reinforcement internal stability.PolyI:C polynucleotides can also be for example, by relying ammonia for molecule and positively charged gathering Acid and carboxymethyl cellulose or with positively charged synthetic peptide is compound is modified for example to enhance it to the resistance degraded in vivo.

In some embodiments, PolyI:C polynucleotides adjuvant is the traditional form of polyI:C, wherein general molecule Amount is 989,486 dalton, the mixture (Thermo of the polyI and polyC of hundreds of base-pairs containing different chain length Scientific；USA).

In some embodiments, composition as disclosed herein may include activation or the increase active adjuvant of TLR2. As used herein, the adjuvant of " activity " of " activation " or " increase " TLR2 includes any adjuvant, in some embodiments, including Adjuvant based on lipid serves as TLR2 agonist.In addition, activation or to increase the activity of TLR2 include it with any monomer, homologous The activation of dimer or heterodimer form, and specifically include TLR2 as the heterodimeric with TLR1 or TLR6 The activation of body (i.e. TLR1/2 or TLR2/6).Activation or the illustrative embodiments for increasing the active adjuvant of TLR2 include base In the adjuvant of lipid, the adjuvant as described in WO2013/049941.

Therefore, in one embodiment, composition as disclosed herein may include the adjuvant based on lipid, such as example Disclosed in WO2013/049941.In one embodiment, the adjuvant based on lipid is PAM₂Cys-Ser-(Lys)4(SEQ ID NO:3) or PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)。

In another embodiment, vaccine composition as disclosed herein may include lipid A mimic or the like Adjuvant, the adjuvant as disclosed in such as WO2016/109880 and references cited therein.In a specific embodiment, it helps Agent can be the JL-265 as disclosed in WO2016/109880 or JL-266.

The further example of workable adjuvant includes being not limited to, chemotactic factor (CF), colony stimulating factor, cell factor, 1018ISS, aluminium salt, Amplivax, AS04, AS15, ABM2, Adjumer, Algammulin, AS01B, AS02 (SBASA), ASO2A, BCG, calcitriol (Calcitriol), chitosan, cholera toxin, CP-870,893, CpG, polyI:C, CyaA, DETOX (RibiImmunochemicals), GERBU Adjuvant 100 (DDA), dibutyl phthalate (DBP), DSLIM, γ inulin, GM-CSF, GMDP, glycerol, IC30, IC31, imiquimod (Imiquimod), ImuFact IMP321, IS Patch, ISCOM, ISCOMATRIX, JuvImmune, LipoVac, LPS, lipid core albumen, MF59, monophosphoryl lipid A With its analog or analogies,IMS1312、Base adjuvant (such as Montanide ISA-51, Montanide ISA-50 and Montanide ISA-70), OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel carry System system, other palmityl molecules, PLG particle, resiquimod (resiquimod), squalene, SLR172, YF-17DBCG, QS21, QuilA, P1005, poloxamer, saponin(e, synthetic polyribonucleotides, zymosan, pertussis toxin.

Therefore, the composition of this paper may include one or more pharmaceutically acceptable adjuvants.In some embodiments, At least one antigen can be coupled at least one adjuvant.

In some embodiments, the composition of this paper may include PolyI:C polynucleotides adjuvant, the assistant based on lipid Agent, lipid A mimic or the like, or combinations thereof.In a specific embodiment, composition may include PolyI:C polynucleotides The combination of adjuvant and the adjuvant based on lipid is such as filed in the U.S. Provisional Patent Application No. 62/256 on November 18th, 2015, Disclosed in auxiliary system disclosed in 875 (adjuvanting system).

The amount of adjuvant used depends on the type of antigen and the type of amount and adjuvant.Those skilled in the art can pass through through Test tries the amount for being readily determined adjuvant needed for concrete application.

In one embodiment, composition as described herein includes lipopeptid adjuvant PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)。

Emulsifier

In some embodiments, compositions disclosed herein may include one or more emulsifying agents.Emulsifier can be The mixture of pure emulsifier or emulsifier.Emulsifier (one or more) should be subjected to pharmaceutically and/or in immunology 's.

The application of emulsifier can have specifically relevant property to not aqueous generally water-free composition is prepared.For example, In some embodiments, when amphipathic compound or mixture are resuspended in hydrophobic carrier, emulsifier can be used for helping to stablize Amphipathic compound, the mixture of amphipathic compound and antigen or amphipathic compound, antigen and other vaccine components (such as PolyI:C and/or adjuvant based on lipid, T auxiliary epitope etc.) mixture.For example, the application of emulsifier can promote amphiphilic Close the distribution of object or mixture in hydrophobic carrier more evenly.

And therefore emulsifier can be amphiphilic, and, emulsifier may include a wide range of compound.In some embodiments In, emulsifier can be surfactant, such as example, nonionic surface active agent.The example of workable emulsifier includes Spheron MD 30/70 (polysorbates) --- it is to glycosylate sorbierite (polyethylene derived from polyethylene Glycolyatedsorbital oil-based liquid) --- and water sorbitol ester.Spheron MD 30/70 may include, for example, Sorbitan Alcohol monoleate (sorbitanmonooleate).Typical emulsifier is well known in the art and including being not limited to, two contractings are sweet Reveal alcohol oleate (Arlacel^TMA), lecithin, Tween^TM 80、Spans^TM20,80,83 and 85.In an embodiment In, it is mannide oleate for the emulsifier in vaccine composition.

Emulsifier is generally pre-mixed with hydrophobic carrier.In some embodiments, it can be used containing emulsifier Hydrophobic carrier.For example, hydrophobic carrier such as (such) Montanide^TMISA 51 has contained emulsifier mannide oleic acid Ester.In other embodiments, hydrophobic carrier can be mixed with emulsifier, then and amphipathic compound；Amphipathic compound and antigen Mixture；Or amphipathic compound, antigen and other vaccine components are (for example, polyI:C and/or adjuvant, T based on lipid are auxiliary Help epitope etc.) mixture combination.

To effectively facilitate amphipathic compound being uniformly distributed in hydrophobic carrier and/or help to form knot as described herein The amount of structure, assembly or array uses emulsifier.Generally, the range of the volume ratio (v/v) of hydrophobic carrier and emulsifier be about 5:1 extremely About 15:1, more specifically 10:1.

T assists epitope

In some embodiments, compositions disclosed herein also may include that at least one T auxiliary epitope or T auxiliary are anti- It is former.This may be especially relevant with the composition comprising the additional antigen with CTL epitope, but as described below, and T assists epitope also Involve (implicated) and is related to the immune response of B cell in mediation.

It is to have the sequence of the amino acid (natural or non-natural amino acids) of T auxiliary activity that T, which assists epitope,.T assists epitope Identified by T helper lymphocyte, establish and maximize immune system ability in play an important role, and be related to activation and Other immunocytes are instructed, such as such as B cell antibody class switch.

T auxiliary epitope can be made of continuously or discontinuously epitope.The not portion of each amino acid necessarily epitope of T auxiliary Point.Therefore, T assists epitope (analog and section including T auxiliary epitope) that can enhance or stimulate immune response.Immundominance T auxiliary epitope is broadly reaction (Celis 1988 in the animal and crowd with MHC type divergent extensively； Demotz1989；Chong1992).The T auxiliary domains of subject peptide (subject peptides) can have about 10 to about 50 Amino acid, and more specifically about 10 to about 30 amino acid.When assisting epitope there are multiple T, then each T auxiliary epitope is only On the spot work.

In some embodiments, T auxiliary epitope can form the part of antigen described herein.Particularly, if antigen has There are enough sizes, then it contains the epitope for being used as T auxiliary epitope.In other embodiments, T assist epitope can be with The molecule of antigen separation.

In another embodiment, it may include 1 to about 10 amino acid in T auxiliary epitope that T, which assists epitope analogs, Substitution, missing and the insertion of residue.T auxiliary section is the adjacent part for being enough to enhance or the T of immune response is stimulated to assist epitope. The example that T assists section is a series of overlapping peptides derived from single longer peptide.

In a specific embodiment, composition as disclosed herein may include that epitope or antigen, modification are assisted as T Tetanus toxin peptide A16L (830 to 844；AQYIKANSKFIGITEL (SEQ ID NO:10), wherein alanine residue is added Its amino terminal is added to enhance stability (Slingluff 2001).

The source that can be used for other T auxiliary epitope of this composition includes, for example, the complementary T of hepatitis B surface antibody Cell epitope, pertussis toxin helper t cell epitope, measles virus F protein helper T lymphocyte epitope, chlamydia trachomatis (Chlamydia trachomitis) major outer membrane protein helper T lymphocyte epitope, diphtheria toxin helper t cell epitope, sickle Shape plasmodial circumsporozoite helper T lymphocyte epitope, schistosoma mansoni triose-phosphate isomerase helper T lymphocyte epitope, large intestine The immune enhancing analog and section of bacillus TraT helper T lymphocyte epitope and any of these T auxiliary epitope.

In some embodiments, T assists epitope to can be general T auxiliary epitope.General T supplementary table as used herein Position refers to peptide or other immunogenic molecules or its segment --- it activates T cell function in a manner of II class (CD4+T cell) limitation The mode of energy combines a variety of (multiplicity of) MHC II class molecules.The example of general T auxiliary epitope is comprising peptide sequence The PADRE (pan-DR epitope) of AKXVAAWTLKAAA (SEQ ID NO:11), wherein X can be cyclohexyl alanyl. Specifically there is PADRE CD4+T to assist epitope, that is, it stimulates the induction of PADRE- specific C D4+ T auxiliary response.

Other than the tetanus toxin peptide A16L of above-mentioned modification, tetanus toxoid have with PADRE class Like mode work (work) other T assist epitope.Tetanus and diphtheria toxin have the general purpose table for people CD4+ cell Position (Diethelm-Okita 2000).In another embodiment, T assists epitope to can be tetanus toxoid peptide, such as wraps (the amino acid 947-967 of FNNFTVSFWLRVPKVSASHLE containing peptide sequence；SEQ ID NO:12) F21E.

In some embodiments, T is assisted in one of epitope and composition as disclosed herein or a variety of antigens At least one fusion (such as fusogenic peptide).

The not aqueous embodiment of composition

In one embodiment, compositions disclosed herein is not aqueous or generally not aqueous, i.e., composition is not cream Agent.

From " not aqueous ", mean that composition is entirely free of water.In another embodiment, composition can be generally It is not aqueous.Term " generally not aqueous " is intended to include the embodiment that wherein hydrophobic carrier still contains a small amount of water, and condition is Water is present in the discontinuous phase of carrier.For example, the individual components of composition can have the technique by being such as lyophilized or evaporating What cannot be completely removed is a small amount of in conjunction with water, and certain hydrophobic carriers can be containing being dissolved in a small amount of water therein.Generally, such as this The composition of " generally not aqueous " disclosed in text contains, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water, the carrier component based on composition total weight (weight/ Weight).Still the composition containing a small amount of water will form emulsion will without containing the water of sufficient amount.

Not by the constraint of any specific function theory, it is believed that when using the not Aquo-composition of the disclosure, preparation creation Strong storage (depot) continues several weeks, allows to extend the removing (clearance) and vaccine and immune system of antigen Interaction.In this regard, it was reported that the preparation based on oily packet rouge reaches peak value clearance rate in 3 weeks of immunization, and And clearance rate continues (Brewer 2014) in 6 months with slow rate.This is with aqueous vaccine preparation or emulsion on the contrary, institute State aqueous vaccine preparation quick release antigen within a few houres to one week；The emulsion forms of short duration storage.

The method for preparing composition

Composition can be by being prepared in view of the means known in the art being originally spaced apart.It retouches below (including in instances) The illustrative embodiments for being used to prepare compositions disclosed herein have been stated, without limiting.

As used in this section, term " antigen " and " adjuvant " are generally used for how description antigen and adjuvant can be prepared in this public affairs In the composition opened.Term " antigen " includes both singular " antigen " and plural form " antigen ".Similarly, term " assistant Agent " includes both singular " adjuvant " and plural form " adjuvant ".All antigens and adjuvant need not be drawn in the same manner Enter into vaccine composition.

In the embodiment for being used to prepare composition, antigen and optionally other components (such as adjuvant, T assist epitope Deng) reconstructed together with amphipathic compound in a suitable solvent.Then dried ingredients are to form dry cake, and dry cake is resuspended to In hydrophobic carrier.Drying steps can be carried out by various means known in the art, such as be steamed by freeze-drying, freeze-drying, rotation Evaporated under hair, certain pressure etc..It also can be used the integrality low-heat for not damaging (compromise) component dry.It can also be used and add Heat is to help resuspension antigen/amphipathic compound mixture.

" suitable solvent " is suitable for the solvent of dissolution antigen, adjuvant and/or amphipathic compound, and can be by technology people Member determines.In one embodiment, sodium phosphate buffer (0.2M, pH 6.0) or sodium phosphate buffer (0.1M, pH can be used 7.0).In another embodiment, polar aprotic solvent can be used, if alcohol is (for example, the tert-butyl alcohol, n- butanol, isopropanol, n- Propyl alcohol, ethyl alcohol or methanol), water, acetate buffer, formic acid or chloroform.In some cases, same solvent dissolution two can be used Then each in close compound, antigen and adjuvant mixes the component of dissolution.Optionally, antigen, adjuvant and amphiphilic chemical combination Object can mix before dissolution, then dissolve together.In further optinal plan, amphipathic compound, antigen or assistant are only dissolved One of agent is a variety of, then adds undissolved component (one or more).

In a specific embodiment, in order to prepare composition, antigen and adjuvant in the phosphorus with S100 lipid and cholesterol It is reconstructed together or separately in sour sodium buffer (Lipoid, Germany).Then these components are lyophilized to form dry cake.Just injecting Before, dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare water-free oil-based composition.

In another embodiment, in order to prepare composition, by antigen and adjuvant in the phosphorus with DOPC and cholesterol It is reconstructed together or separately in sour sodium buffer (Lipoid, Germany).Then these components are lyophilized to form dry cake.Just injecting Before, dry cake is resuspended in ISA51 VG oil (SEPPIC, France) to prepare water-free oil-based composition.

In another embodiment, in order to prepare composition, antigen and synthesis lipid/cholesterol nanoparticle is (big Small≤110nm) and adjuvant mixing in sodium phosphate buffer (100mM, pH 6.0).Synthesis lipid can be DOPC.Then will Component is lyophilized to form dry cake.Just before injection, dry cake is resuspended in ISA51 VG oil (SEPPIC, France) with preparation Water-free oil-based composition.

It, can be by the way that antigen be dissolved in 10%DMSO/0.5% acetic acid aqueous solution (w/ in each above embodiment W) it is heated overnight in mixture and at 37 DEG C, by the preparatory dimerization of antigen.

In a specific embodiment, composition can be prepared by following step:

By antigen being dissolved in the mixture of 10%DMSO/0.5% acetic acid aqueous solution (w/w) and being added at 37 DEG C Heat overnight, prepares the antigen of dimerization；

The lipid mixture being sized is prepared with synthesis lipid/cholesterol nanoparticle (size≤110nm)；

By the antigen of dimerization and synthesis lipid/cholesterol nanoparticle and adjuvant in sodium phosphate buffer (100mM, pH 6.0) mixing in；

Mixture is lyophilized to form dry cake；And

The dry cake of resuspension in hydrophobic carrier.

In embodiment of above, in the case where not fettered by specific function theory, it is believed that the removal (drying) of solvent Stay in component (including antigen) in the array of (leaves) amphipathic compound molecule, wherein its hydrophilic head base is towards vaccine component Orientation.Then vaccine component and amphipathic compound can be suspended in the absence of water in hydrophobic carrier (such as oil), because Keep them sufficiently hydrophobic.

Annexing ingredient (such as T assists epitope) as described herein can be in process for preparation (formulation process) Any stage addition.For example, can by one or more this annexing ingredients before or after dissolution with antigen, adjuvant and/ Or amphipathic compound combination, or it is added to the mixture of dissolution.In another embodiment, alternatively annexing ingredient is added Be added to the drying composite or in combination or combine with hydrophobic carrier of antigen, adjuvant and amphipathic compound --- by antigen, Before or after the dry mixture of adjuvant and amphipathic compound is resuspended in hydrophobic carrier.In one embodiment, T is auxiliary Epitope is helped to be added in composition with antigen same way.In one embodiment, antigen and T auxiliary epitope are fusions Peptide.

It in some embodiments, can the group appropriate that help to stablize dry cake comprising emulsifier in hydrophobic carrier Point --- when it is resuspended in hydrophobic carrier.To be enough the dry mixture resuspension of antigen, adjuvant and amphipathic compound In hydrophobic carrier and antigen, adjuvant and amphipathic compound are maintained at the amount in hydrophobic carrier with suspended pattern, emulsification is provided Agent.For example, emulsifier can exist with about 5% to about 15% w/w or weight/volume of hydrophobic carrier.

It maintains bioactivity or improves chemical stability to extend the steady of the pot-life (shelf life) of any component Composition can be added to by determining agent (such as sugar, antioxidant or preservative).

Immune response and treatment indication

This disclosure relates to utilize the composition comprising the antigen containing B cell epitope as described herein of low dosage volume The method of induction of antibodies immune response, corresponding application and the composition and reagent that can be used for this method in the experimenter Box.

As mentioned in this article, unless otherwise specified, otherwise expresses " immune response " and refer to antibody mediated immunity response.

It is immunized with cell-mediated on the contrary, " antibody mediated immunity response " or " humoral immune response " (interchangeably make herein With) it is, the antibody of the secretion in bone-marrow-derived lymphocyte lineage (B cell) generation antibody-mediated by what is secreted.This secretion Antibody and antigen binding, such as such as foreign matter, pathogen (such as virus, bacterium) and/or the surface of cancer cell on it is anti- Original, and indicate (flag) they for destroying.

As used herein, " humoral immune response " refers to antibody tormation and can also additionally or altematively include with it Attached procedure (accessory processes), such as such as t helper cell 2 (T-helper 2, Th2) or t helper cell 17 The generation and/or activation of (T-helper 17, Th17), cell factor generate, isotype is converted, affinity maturation and memory are thin Born of the same parents' activation." humoral immune response " may also include the effector function of antibody, and such as such as toxin neutralizes, classical complement activation is made With and the promotion eliminated of phagocytosis and pathogen.Humoral immune response must usually help CD4+ Th2 cell, and therefore should The activation or generation of cell type can also indicate that humoral immune response.Term " humoral immune response " herein can be with " antibody Response " or " antibody mediated immunity response " are used interchangeably.

" antibody " is comprising substantially or partly by immunoglobulin gene or the fragment coding of immunoglobulin gene One or more polypeptides protein.The immunoglobulin gene of identification includes κ, λ, α, γ, δ, ε and μ constant region gene, with And myriad immunoglobulin variable region gene.Light chain is classified as κ or λ.Heavy chain is classified as γ, μ, α, δ or ε, and then point Not Xian Ding immunoglobulin class, IgG, IgM, IgA, IgD and IgE.Typical immunoglobulin (antibody) structural unit contains There are four the protein of polypeptide.Each antibody structural unit is made of two pairs of identical polypeptide chains, all have " light " chain and One " weight " chain.The end N- of every chain limits the variable region for being mainly responsible for antigen recognizing.(such as IgA and IgM class) antibody Structural unit can be assembled in the form of mutual oligomerization with Additional polypeptides chain, such as IgM pentamer in conjunction with J chain polypeptide.

Antibody is known as the antigentic specificity glycoprotein product of the leukocyte sub-type of bone-marrow-derived lymphocyte (B cell).Antigen with The engagement for the antibody expressed on B cell surface can induce antibody response, including stimulation B cell undergoes mitosis to be activated And finally breaking up plasmablast, thick liquid cell is dedicated for synthesis and secretion antigen specific antibody.

Unique producer of antibody during B cell is immune response, therefore be the key element of effective humoral immunity.In addition to Generate except a large amount of antibody, B cell act also as antigen-presenting cells and can by antigen presentation to T cell, as T assist CD4 or Cytotoxicity CD8+ T cell, to conduct (propagating) immune response.B cell and T cell are that adaptive immunity is answered The part answered.During the active immunity response for example induced by inoculation or natural infection, antigen-specific b cells are activated And it expands with cloning.During expansion, B cell development is to have higher compatibility to epitope.The proliferation of B cell can be by swashing T helper cell indirect induction living can also directly be induced by the stimulation of receptor (such as TLRs).

Antigen presenting cell, such as dendritic cells and B cell, be attracted to inoculation position and can with contain in vaccine composition Some antigen and adjuvant interaction.Generally, adjuvant stimulation cell is to be allowed to be activated and antigen as target provides blueprint (blueprint).Different types of adjuvant can provide different stimulated signal to cell.For example, poly I:C (TLR3 agonist) Dendritic cells can be activated, but B cell cannot be activated.Adjuvant (such as Pam3Cys, Pam2Cys and FSL-1) is especially good at activation and is opened The proliferation of dynamic B cell, it is contemplated that it promotes the generation (Moyle 2008 of antibody response；So 2012).

Humoral immune response be one of common mechanism of effective infectious diseases vaccine (such as with for virus or bacterium invade Enter object to be protected).However, humoral immune response can also be used for anticancer.Although cancer vaccine is generally designed to generate It can recognize and destroy the cell-mediated immune response of cancer cell, but the response that B cell mediates can be by for maximum benefit Place can in some cases with other mechanism target on cancer cells of cytotoxic T cell cooperation.B cell mediation (such as body What liquid immune response mediated) example of antitumor response includes being not limited to: 1) by be integrated to tumour cell or influence tumour hair The antibody that the B cell of the surface antigen found on raw other cells generates.This antibody can be for example thin by antibody dependent The killing of cytotoxicity (ADCC) or complement zygotic induction target cell that born of the same parents mediate, potentially resulting in can be by immune system identification The release of additional antigens；2) receptor on tumour cell is integrated to block their effect for stimulating and actually neutralizing them Antibody；3) to pass through the relevant factor knot of cell tumour or tumour relevant cell release or relevant with tumour or tumour It closes, to adjust the antibody of the signal transduction or cellular pathways of supporting cancer；And it is 4) current with intracellular target knot merga pass The antibody of unknown mechanisms mediate anti-tumor activity.

A kind of method of assessment antibody response is the potency for the antibody that measurement is reacted with specific antigen.This is using this field Known various methods (enzyme linked immunosorbent assay (ELISA) (ELISA) of substance antibody-containing is such as obtained from animal) carry out.For example, The potency for being integrated to the serum antibody of specific antigen can measure in subject before and after being exposed to antigen.It is exposed to anti- The statistically significant increase of the potency of antigen-specific antibodies after original will indicate that (mounted) is unfolded to antigen in subject Antibody response.

Without limitation, can be used for detecting existing for antigen-specific antibodies it is other measurement include immunologic assay (such as Radiommunoassay (RIA)), immune precipitation determination and protein imprinted (such as Western marking) measurement；And it neutralizes Measurement (such as neutralizing viral infection in measuring in vitro or in vivo).

Method described herein and composition can be used to treat or prevent the disease and/or disease improved by humoral immune response Disease.Method and composition can give antigen to subject any desired to be answered in the case where induction body fluid immune response With.

In some embodiments, method disclosed herein can be used for treating and/or preventing by bacterium, virus, fungi, post Biology, allergen express disease caused by the tumour cell of antigen.

As used herein, " treatment (treating) " or " treatment (treatment of) " or " prevention (preventing) " or " prevention (prevention of) " refers to the scheme for obtaining beneficial or desired result.It is beneficial Or desired result may include but being not limited to, the alleviation or improvement of one or more symptoms or the patient's condition, disease degree subtract Gently, the prevention of stabilization, the disease development of morbid state, the prevention of transmission, the delay of progression of disease or slow down (such as press down System), the delay of seizure of disease or slow down, assign protective immunity and disease for pathogenic agent (disease-causing agent) The improvement or mitigation of diseased state.The time-to-live (survival) that " treatment " or " prevention " also can refer to extend patient is more than not control The expected time-to-live in the case where treatment, and also can refer to the progress for temporarily inhibiting disease or the generation for preventing disease, it is such as logical Cross the infection in prevention subject." treatment " also can refer to the reduction of tumor mass (tumor mass) size, tumor invasiveness Reduce etc..

" treatment " can be different from " prevention " because " treatment " typically occur in it is having suffered from disease or illness or known Be exposed in the subject of infectious agent (infectious agent), and " prevention " typically occur in do not suffer from disease or In illness the or unknown subject for being exposed to infectious agent.It will be understood that treating and preventing, there may be overlappings.Example Such as, it is possible to which the disease of " treatment " subject " prevents " symptom or progress of disease simultaneously.In addition, at least above and below inoculation Wen Zhong, " treatment " and " prevention " can be overlapped, because the treatment of subject will induce immune response, which can have pre- The anti-subsequent effect of potential disease or symptom caused by pathogenic infection by pathogenic infection or prevention.These prevention aspects Improving the statement for such as " treating infectious diseases " herein is included.

In one embodiment, method disclosed herein and composition can be used for treating and/or preventing in the experimenter Such as the infectious diseases as caused by virus infection.Subject may be infected or be likely to be at the wind for developing virus infection Danger.

The virus infection that can be treated and/or prevent by low dosage volume method disclosed herein includes being not limited to, cowpox Virus, vaccinia virus, pseudocowpox virus, 1 type of nerpes vinrus hominis, 2 type of nerpes vinrus hominis, cytomegalovirus, mankind's adenopathy Malicious A-F type, polyomavirus, human papilloma virus (HPV), parvovirus, hepatitis A virus, hepatitis type B virus, the third type Hepatitis virus, human immunodeficiency virus, positive reovirus genus, rotavirus, Ebola virus, parainfluenza virus, A type stream Influenza Virus, influenza B virus, influenza virus C, measles virus, mumps virus, rubella virus, Pneumovirus, Ren Leihu Inhale road syncytial virus, hydrophobin, california antigenic group viruses, japanese encephalitis virus, hantaan virus, lymphatic Choriomeningitis virus, coronavirus genus, enterovirus genus, Rhinovirus, poliovirus, norovirus, jaundice Malicious category, dengue fever virus, West Nile Virus, flavivirus and varicella.In a specific embodiment, virus infection is human milk Head tumor virus, Ebola virus, human airway syncytial virus or influenza virus.

In one embodiment, method disclosed herein can be used for treating and/or preventing the lower respiratory tract in the experimenter Infection, wherein infection is caused by respiratory syncytial virus (RSV) (RSV).In one embodiment, infection is sub- by RSV A Caused by group's Strain.In one embodiment, infection is as caused by RSV B subgroup virus strain.

In another embodiment, method disclosed herein or composition can be used for treating and/or preventing such as to it The infectious diseases as caused by non-viral pathogen (such as bacterium or protozoan) in the experimenter in need.Subject can quilt Pathogenic infection is likely to be at the risk for developing pathogenic infection.Without limitation, exemplary bacterial pathogens may include charcoal Subcutaneous ulcer (bacillus anthracis), Brucella, pertussis Bao Te bacillus, Mycotoruloides, chlamydia pneumoniae, chlamydia psittaci, cholera, Clostridium botulinum, posadasis spheriforme, Cryptococcus, diphtheria, Escherichia coli O 157: H7, enterohemorrhagic escherichia coli, intestines Enterotoxigenic E. Coli, haemophilus influenzae, helicobacter pylori, Legionnella, Leptospira, Li Site Pseudomonas, meninx Scorching coccus, mycoplasma pneumoniae, Mycobacterium, pertussis, pneumonia, Salmonella, Shigella, staphylococcus, pneumonia streptococcus Bacterium and yersinia enterocolitica.In a specific embodiment, bacterium infection is anthrax.Without limitation, exemplary primary Animal pathogen may include Plasmodium (plasmodium falciparum, malariae, Plasmodium vivax, the ovale malaria original for causing malaria Those of worm or Plasmodium knowlesi).

In one embodiment, method disclosed herein and composition can be used for treating in the experimenter in need thereof Cancer.In one embodiment, cancer is the cancer for the film surface combination cancer antigen that expression can be identified by antibody.

As used herein, term " cancer ", " cancer cell ", " tumour " and " tumour cell " (being interchangeably used) refers to Excrescent cell is shown it is characterized in that significantly losing the control of cell proliferation) or immortalized thin Born of the same parents.Term " cancer " or " tumour " include metastatic and non-metastatic cancer or tumour.Cancer can generally be connect using this field The standard diagnostics received, the presence including pernicious tumour.

Without limitation, by using or give the cancer that composition as disclosed herein can treat and/or prevent Including cancer (carcinoma), gland cancer, lymthoma, leukaemia, sarcoma, blastoma, myeloma and gonioma.It is unrestricted Property, specially suitable embodiment may include spongioblastoma, Huppert's disease, oophoroma, breast cancer, fallopian tubal Cancer, prostate cancer or peritoneal cancer.In one embodiment, cancer can be caused by pathogen (such as virus).With cancer development phase The virus of connection is known for the skilled person and includes, but are not limited to human papilloma virus (HPV), JC virus (John Cunningham virus, JCV), human herpes virus 8, angstrom bar virus (EBV), Merkel cell polyomavirus, the third type Hepatitis virus and human T cell leukemia virus -1.In another embodiment, cancer can be the one or more cancers of expression The cancer of disease-specific antigen (such as survivin).

In a specific embodiment, cancer is breast cancer, oophoroma, prostate cancer, carcinoma of fallopian tube, peritoneal cancer, collagen Cytoma or diffusivity large B cell lymphoid tumor.

In another embodiment, method disclosed herein or composition can be used for treating and/or preventing having it needing The neurodegenerative disease in subject wanted, wherein neurodegenerative disease is related with the expression of antigen.Subject can be with nerve Degenerative disease or the risk that can be at developing neurodegenerative disease.Can by method disclosed herein or composition treatment and/or The neurodegenerative disease of prevention includes being not limited to, Alzheimer disease, Parkinson's disease, Huntington disease and amyotrophic lateral sclerosis (ALS)。

In another embodiment, method disclosed herein or composition can be used for treating and/or preventing additive disease Sick (e.g., for example, to ***e habituation).

In another embodiment, method disclosed herein or composition can be used for neutralizing a toxin using antibody, virus, Bacterium or allergen, the method includes giving composition as described herein to subject.For example, in response to anti-in vaccine The former and antibody that generates can in and/or isolation toxin, virus, bacterium or allergen.In one embodiment, toxin is drug, As for example, ***e.

Kit (set group, kits), combination and reagent

Compositions disclosed herein is provided as kit to user.For example, the kit of the disclosure includes One or more components of compositions disclosed herein.Kit can further comprise one or more additive reagents, packing timber It material, container for accommodating kit components and is described in detail and illustrates external member using the preferred method of kit components (instruction set) or user's manual.In one embodiment, container is bottle (vials).

In one embodiment, kit includes in a reservoir the vaccine prepared in advance with available form at any time.As One example, in one embodiment, kit include at least one appearance comprising amphipathic compound, antigen and hydrophobic carrier Device.The vaccine prepared in advance can further include adjuvant or any other annexing ingredient.

In the alternative embodiments of kit, other than hydrophobic carrier, vaccine can be mentioned together with all components For in a vessel (such as dry cake), in case reconstructed in hydrophobic carrier, or it is provided at point as single component In the container opened, in case preparing, being lyophilized and reconstructing in hydrophobic carrier.

In one embodiment, kit may include the first container comprising amphipathic compound and antigen；With comprising dredge The second container of water carrier.In this embodiment, the vaccine component in the first container may be at the form of dry cake, prepare weight It is suspended in hydrophobic carrier.

In one embodiment, kit may include (i) to dry in the form of freeze-dried powder comprising antigen, adjuvant and phosphatide First bottle, and (ii) include the second bottle that hydrophobic carrier is used as diluent.

In the various aspects of mentioned reagent box embodiment, other than antigen, amphipathic compound and hydrophobic carrier, epidemic disease Seedling is also optionally including one or more adjuvants, T auxiliary epitope and emulsifier.These components can be provided separately in separated It in container or can be provided in container together with any combination thereof, appearance is such as provided in together with antigen and amphipathic compound In device.

In the embodiment of kit, antigen is peptide antigen, and it includes A subgroup or the small hydrophobic eggs of RSV plants of B subgroup people White extracellular domain (SHe).For example, in one embodiment, antigen includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1) is made of it.

In the embodiment of kit, adjuvant is PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)。

In the embodiment of kit, amphipathic compound is one or more lipids, such as phosphatide.In an embodiment In, lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol.

In the embodiment of kit, hydrophobic carrier isISA 51 VG。

Therefore, in one embodiment, kit includes two bottles, and wherein content is as follows in detail:

The skilled person will understand that the alternative configuration (arrangements) of kit is possible, and include by the disclosure.

Kit as disclosed herein can be used for practicing method disclosed herein.In one embodiment, kit For the antibody mediated immunity response for antigen to be induced in the experimenter using the composition of the low dosage volume in kit.One In a embodiment, kit is used to prepare not aqueous or generally water-free composition.

Embodiment

The specific embodiment of the disclosure includes being not limited to, the following contents:

(1) method for the induction of antibodies immune response in the experimenter, including low dose is parenterally given to the experimenter The composition of volume is measured, the composition includes:

Antigen containing B cell epitope；

Amphipathic compound；With

Hydrophobic carrier,

Wherein the low dosage volume of composition induces the antibody for B cell epitope to exempt from less than 100 μ l and in the experimenter Epidemic disease response.

The method of (2) (1) moneys, wherein low dosage volume is about 50 μ l, about 60 μ l, about 70 μ l, about 80 μ l or about 90 μ l.

The method of (3) (1) moneys or (2) money, wherein low dosage volume is in about 50 μ l between about 75 μ l.

(4) (1) moneys are to the method for any one of (3) money, and wherein low dosage volume is about 50 μ l.

(5) (1) moneys to the method for any one of (4) money, wherein composition include about 5 μ g, about 10 μ g, about 15 μ g, The antigen of about 20 μ g, about 25 μ g, about 30 μ g, about 35 μ g, about 40 μ g, about 45 μ g or about 50 μ g.

The method of (6) (5) moneys, wherein composition includes the antigen of about 10 μ g or about 25 μ g.

(7) (1) moneys to any one of (6) money method, including be given only it is single for the first time give with it is single reinforce to The composition given.

The method of (8) (7) moneys, wherein it is single reinforcement give it is single for the first time give after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks or longer time mentioned to the experimenter For.

The method of (9) (7) moneys or (8) money, wherein it is single reinforcement give it is single for the first time give after about 56 days by The experimenter provides.

(10) (1) moneys are given to the method for any one of (6) money including being given only the single of composition.

(11) (1) moneys further comprise giving low dosage body at least once to the method for any one of (10) money After long-pending composition, the antigen with post dose is given to the experimenter, the antigen is not to include hydrophobic carrier or not include hydrophobic The water-based composition of carrier and amphipathic compound is prepared.

(12) (1) moneys to any one of (11) money method, wherein being induced by the composition of low dosage volume anti- Body immune response is detectable at least as early as giving after composition 28 days for the first time in the experimenter.

(13) (1) moneys to any one of (12) money method, wherein being induced by the composition of low dosage volume anti- Body immune response continues at least 84 days after giving composition for the first time.

(14) (1) moneys to any one of (13) money method, wherein being induced by the composition of low dosage volume anti- Body immune response continues at least 236 days after giving composition for the first time.

(15) (1) moneys are to the method for any one of (14) money, and wherein the composition of low dosage volume is after giving 56 days, the 84th day or the 236th day at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% The treated experimenter in induction of antibodies immune response.

The method of (16) (15) moneys, wherein the composition of low dosage volume is at least the 84th day or the 236th day after giving The induction of antibodies immune response in the 100% treated experimenter.

(17) (1) moneys to any one of (16) money method, wherein with to give alum composition prepare antigen The experimenter in the antibody mediated immunity response that induces compare, increased by the antibody mediated immunity response that the composition of low dosage volume induces By force, wherein alum composition includes antigen, alum adjuvant, mineral adjuvant (such as Alhyrogel) and aqueous carrier.

The method of (18) (17) moneys, wherein the enhancing of antibody mediated immunity response is that have for the detectable antigen spy of antigen The increase of the percentage of the experimenter of heterogenetic antibody immune response and/or the increase of mean antigen specific antibody endpoint titers.

The method of (19) (18) moneys, wherein every kind in the composition and alum composition includes the antigen of 10 μ g, with And mean antigen specific antibody endpoint titers in the case where the composition: (i) was combined at the 28th day after giving than alum Up at least about 123.2 times of object；And/or (ii) at the 56th day than up at least about 16.7 times of alum composition.

The method of (20) (18) moneys, wherein every kind in the composition and alum composition includes the antigen of 25 μ g, with And the mean antigen specific antibody endpoint titers of the composition: (i) is higher than alum composition at least at the 28th day after giving About 35.3 times；And/or (ii) at the 56th day than up at least about 13.1 times of alum composition.

The method of (21) (18) moneys, wherein every kind in the composition and alum composition includes the antigen of 10 μ g, with It is tested with detectable antigen-specific antibodies immune response when being treated with the composition and the 56th day after giving Up at least about 2.0 times when the percentage ratio alum composition of people.

The method of (22) (18) moneys, wherein every kind in the composition and alum composition includes the antigen of 25 μ g, and It is tested with detectable antigen-specific antibodies immune response when being treated with the composition and the 56th day after giving Up at least about 5.0 times when the percentage ratio alum composition of people,.

(23) (17) moneys to any one of (22) money method, wherein the whole body adverse events about overall demand And/or the adverse events of overall non-demand, composition have acceptable safety (safety profile).

(24) (1) moneys are to the method for any one of (23) money, and wherein the experimenter is baby, teenager, adult or old Year subject.

The method of (25) (24) moneys, wherein the experimenter has 0-2 years old, 50-64 years old or the age more than 65 years old.

(26) (1) moneys are to the method for any one of (25) money, and wherein composition further includes adjuvant.

The method of (27) (26) moneys, wherein adjuvant is PolyI:C polynucleotides adjuvant, the adjuvant based on lipid, lipid A Analogies or its analog, or any combination thereof.

The method of (28) (27) moneys, wherein the adjuvant based on lipid is lipopeptid, such as PAM₂Cys-Ser-(Lys)4 (SEQ ID NO:3) or PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)。

(29) (1) moneys are to the method for any one of (28) money, and wherein antigen is the peptide that length is 5 to 50 amino acid The polynucleotides of antigen or encoded peptide antigen.

(30) (1) moneys are to the method for any one of (29) money, and wherein antigen is that have naturally weak exempt from the experimenter The antigenic synthetic peptide of epidemic focus.

(31) (1) moneys are to the method for any one of (30) money, wherein antigen: (i) is derived from virus, bacterium or primary Animal, such as example Ebola virus, zika virus, human papilloma virus (HPV), influenza virus, respiratory syncytial virus (RSV), Pertussis Bao Te bacillus, bacillus anthracis or malariae；It (ii) is film surface combination cancer antigen, such as example survivin is anti- It is former；It (iii) is toxin, such as such as ***e；Or (iv) is neoantigen.

(32) (1) moneys are to the method for any one of (31) money, and wherein antigen includes the small hydrophobin (SH) of virus Its segment or by virus small hydrophobin (SH) or its segment constitute.

The method of (33) (32) moneys, wherein virus is paramyxovirus.

The method of (34) (33) moneys, wherein paramyxovirus is respiratory syncytial virus (RSV) (RSV), such as people RSV.

(35) (32) moneys are to the method for any one of (34) money, and wherein antigen includes the extracellular domain (SHe) of SH Or it its segment or is made of the extracellular domain (SHe) of SH or its segment.

The method of (36) (35) moneys, wherein SHe is derived from RSV plants of RSV plants of A subgroup people or B subgroup people.

The method of (37) (36) moneys, wherein SHe includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1) or NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) or same with SEQ ID NO:1 or 2 at least 75% The amino acid sequence in source.

The method of (38) (37) moneys, wherein antigen includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1)、NKLSEYNVFHNKTFELPRARVNT(SEQ ID NO:6)、NKLSEHKTFCNKTLEQGQMYQINT(SEQ ID NO:2) or its segment or by amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1), NKLSEYNVFHNKTFELPRARVNT (SEQ ID NO:6), NKLSEHKTFCNKTLEQGQMYQINT (SEQ ID NO:2) or Its segment is constituted.

The method of (39) (38) moneys, wherein antigen is by amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1 it) constitutes.

(40) (35) moneys are to the method for any one of (39) money, and wherein SHe exists with oligomer such as dimer.

(41) (35) moneys are to the method for any one of (40) money, and wherein SHe connects on gene or chemically with carrier It connects.

The method of (42) (41) moneys, wherein carrier is amphipathic compound or the structure that is formed by it.

(43) (31) moneys are included in the experimenter and are exposed to virus, bacterium, primary to the method for any one of (42) money Before animal or toxin, the composition of low dosage volume is given to the experimenter.

(44) (1) moneys are to the method for any one of (43) money, and wherein antigen is sufficiently hydrophobic or is made sufficiently to dredge Water, so that antigen can mix in hydrophobic carrier.

The method of (45) (44) moneys, wherein keeping antigen sufficiently hydrophobic by the presence of amphipathic compound in composition.

(46) (1) moneys are to the method for any one of (45) money, and wherein amphipathic compound is lipid or lipid mixture.

The method of (47) (46) moneys, wherein lipid or lipid mixture include phosphatide, such as phosphatidyl choline, dioleoyl Or mixtures thereof base phosphatidyl choline (DOPC), lecithin (such as Lipoid S100).

The method of (48) (46) moneys or (47) money, wherein amphipathic compound is dioleyl phosphatidyl choline (DOPC) With the lipid mixture of cholesterol.

(49) (46) moneys are to the method for any one of (48) money, and wherein lipid is formed partly or completely around anti- Former piece or balloon-shaped structure.

(50) (46) moneys are to the method for any one of (48) money, and wherein lipid forms closing blister knot around antigen Structure, such as single layer balloon-shaped structure (such as micella) or the double-deck balloon-shaped structure (such as single-layer or multi-layer liposome).

(51) (1) moneys are to the method for any one of (50) money, and wherein hydrophobic carrier is the mixture of oil or oil.

The method of (52) (51) moneys, wherein hydrophobic carrier include vegetable oil, macadamia nut oil, mineral oil, or mixtures thereof.

The method of (53) (52) moneys, wherein hydrophobic carrier is the mineral oil of mineral oil either mannide oleate Solution, such asISA 51 VG。

(54) (1) moneys to the method for any one of (53) money, wherein composition includes:

Peptide, it includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO:1)；

Lipid mixture, it includes dioleyl phosphatidyl choline (DOPC) and cholesterol；

Short synthesis lipopeptid, is PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)；And

ISA 51 VG。

(55) (1) moneys are to the method for any one of (54) money, and wherein composition is not aqueous or generally not aqueous.

The method of (56) (55) moneys, wherein generally water-free composition include less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water, the total weight based on carrier (w/w).

Method of (57) (1) moneys to any one of (56) money, the combination including giving low dosage volume by injection Object.

The method of (58) (57) moneys, the composition including intramuscular injection low dosage volume.

(59) (1) moneys are used to treat or prevent the infectivity in the experimenter to the method for any one of (58) money Disease.

(60) composition of low dosage volume is for the answering for B cell epitope induction of antibodies immune response in the experimenter With the composition includes:

Antigen comprising B cell epitope；

Amphipathic compound；And

Hydrophobic carrier, wherein composition is given for parenteral and the low dosage volume of composition is less than 100 μ l.

(61) according to the application of (60) money, in which: low dosage volume limits in any one of (2) money to (4) money It is fixed；The amount of antigen limits in (5) money or (6) money；Composition is with (7) money to (11) money, (43) money, (57) For giving defined in any one of money and (58) money；Any of antibody mediated immunity response in (12) money to (22) money It is limited in；Using safety defined in offer (23) money；The experimenter limits in (24) money or (25) money；Group It closes object and further includes the adjuvant that (26) money is limited into any one of (28) money；Antigen is in (29) money to (42) It is limited in any one of money, (44) money and (45) money；Amphipathic compound is in any one of (46) money to (50) money It limits；Hydrophobic carrier limits in any one of (51) money to (53) money；And/or composition is in (54) money to (56) It is limited in any one of money.

(62) according to the application of (60) money or (61) money, it is used to treat or prevent the infectious disease in the experimenter Disease, such as treat or prevent RSV.

(63) it is used for the compositions or agents box of the method according to (1) money to any one of (59) money.

The present invention is further illustrated by the following non-limiting examples.

Embodiment

Clinical trial protocol

1 phase studied (refering in particular to CI1204) and carries out (NCT#02472548) in Canada, had evaluated what low dosage volume was given Oil-based formulation is used to be directed to respiratory syncytial virus (RSV) (RSV) small hydrophobic extracellular domain A type (SHe A) peptide antigen induced immune Safety of the response compared to alum based formulation and immune efficacy.Research is randomization, the blind (observer- of observer Blind), placebo, dosage escalation (increase, escalation) test.Research is related to two steps, and the first step is commented Estimate low peptide dosage particles, second step assesses high peptide dosage particles.

RSV SHe A peptide antigen (NKLCEYNVFHNKTFELPRARVNT used in the research；SEQ ID NO:1) by PolyPeptide (San Diego, USA) synthesis.By the way that RSV SHe A peptide antigen is dissolved in 10%DMSO/0.5% acetic acid (10 mg/ml) and heated at 37 DEG C make peptide thioether yesterday in the mixture of aqueous solution (w/w).It is used in the research Oil base vaccine be made of the RSV SHe A peptide antigen of 0.2 mg/ml (low peptide) or 0.5 mg/ml (high peptide).Low peptide It further include the lipopeptid adjuvant (PAM of 0.04 mg/ml with both high peptide oil base vaccines₃Cys-Ser-(Lys)4；SEQ ID NO: 3；PolyPeptide, San Diego, USA), the synthesis 1,2-dioleoyl-sn-glycerol base -3- phosphoric acid of 120 mg/mls Choline lipid (DOPC；Lipoid, GmbH, Germany) and 12 mg/mls cholesterol (Lipoid, GmbH, German).Vaccine exists Delivering in 51 VG of ISA oil (SEPPIC, France).In order to prepare oil base vaccine, the RSV SHe A peptide antigen of dimerization and synthesis Lipid/cholesterol nanoparticle (size≤110nm) and lipopeptid adjuvant are in sodium phosphate buffer (100 mMs, pH 6.0) (in the form of high peptide or low peptide) mixing.Then vaccine component is lyophilized to form dry cake.Just before injection, by dry cake resuspension In oil.

The composition of reconstruct is as follows:

This research used in alum base vaccine by 0.2 mg/ml (low peptide) or 0.5 mg/ml (high peptide) RSV SHe A peptide antigen is constituted.Both low peptide and high peptide alum base vaccine also include2%, 10 milligrams of aluminium/milliliters (Brenntag Biosector A/S, Denmark).In order to prepare alum base vaccine, the RSV SHe A peptide antigen second of dimerization (10 mMs, pH 7.0) of sour sodium buffer dilute and fill into bottle.Just before injection, by vaccine with2% (high peptide) --- 1 milligram of aluminium/milliliter vaccine product --- or2%/water (low peptide) Mixing.

The alum composition of reconstruct is as follows:

The subject for participating in clinical test is the health adult between 50-64 years old age.Average age is 55.3 years old, and 72.5% is women.Four groups of subjects (n=8) are inoculated with as follows: 1) 50 microlitres of low peptide oil-based formulation, in research the 0th day and the 56 days；2) 50 microlitres of low peptide alum based formulation, in research the 0th day, then 50 microlitres of saline placebo, was studying the 56th It；3) 50 microlitres of high peptide oil-based formulation, in research the 0th day and the 56th day；And 4) 50 microlitres of high peptide alum based formulation, It studies the 0th day, then 50 microlitres of saline placebo, is being studied the 56th day.5th group of subject (n=8) research the 0th day and Receive within 56th day the saline placebo (0.9% sodium chloride) of 50 microlitres of dose volumes.

Compare agent group (comparator groups) includes that (placebo and RSV (A)-alum) allows to estimate not Good event can attribution risk.The research of safety review board (Safety Review Committee, SRC) keeps rule (Study holding rules) and security evaluation are in place.

Embodiment 1- safety and reactionogenicity

Pass through demand and non-demand Adverse event monitoring and measurement safety.Study the 0th day first time give and It is given for the 56th day second in research, then every day entry event duration 1 week.

There is no serious adverse events to report.The local adverse events of the most common demand of report are injection site pains.Not yet There is the report of local redness or swelling.Importantly, the delivering of the oil-based composition with booster, does not observe injection part The increase of position pain.

The whole body adverse events of the most common demand of report are drowsiness, nausea, diarrhea and myalgia.For overall non-demand Adverse events do not report serious adverse events, and restore or solve all other event.

In general, as a result indicate that the oil base of low dosage volume and alum based formulation have acceptable safety, and There is no serious safety consideration.

Embodiment 2- immunogenicity

It is collected by the blood serum of regular intervals of time under study for action, vaccine is monitored to all subjects in clinical test Immunogenicity.It is detected for the antibody of RSV SHe Staphylococal Protein A by enzyme linked immunosorbent assay (ELISA) (ELISA).

In brief, RSV SHe A peptide dimer used in 96 orifice plate clinical vaccines is with 1 mcg/ml (in carbon In phthalate buffer (pH 9.0)) it is coated with 18-21 hours at 4 DEG C.Then by phosphorus of the plate with 0.05% tween (PBS-T) It is small that (blocked) 1 is closed in hydrochlorate buffered saline wash and at room temperature use measurement diluent (ChonBlock, Chondrex) When.The serial dilution (Serial dilutions) of serum is prepared in measurement diluent and is added after being washed with PBS-T To plate.The 0th day serum of each plate testing research is repeated 5 times and other time point is triplicate.Plate is incubated at room temperature 2.5-3 Hour, then washed with PBS-T.The anti-human igg of peroxidase labelling is added in hole, and plate is incubated at room temperature It 1 hour, is then washed with PBS-T and PBS.TMB microwell peroxidase (peroxdisase) substrate system is conjugated in enzyme (KPL) it is added in hole, and allows to develop at room temperature up to 4 minutes, then adds TMB terminate liquid (KPL).In ASYS The optical density in hole is read on Expert Plus Microplate Reader at 450nm.According to the method for Frey A. et al. (Journal of Immunological Methods, 1998,221:35-41), the 0th day OD of use research calculate OD critical value (cutoff).The potency of calculating indicates to detect the increased highest dilution of OD relative to critical value.It is dilute that potency is expressed as terminal The log10 value of degree of releasing inverse.The detection limit of the measurement is 1/100 dilution, and the response lower than the limit is reported as log 10 =0.

The endpoint titers for receiving the subject of low peptide vaccine are shown in Fig. 1.Receive the terminal of the subject of high peptide vaccine Potency is shown in Fig. 2.Table 1 summarizes the endpoint titers of the group with low or high peptide oil and the inoculation of alum based formulation.

Table 1: for mean end point titre of the subject through SEM in oil base or alum base vaccine group

In research the 56th day, all groups received single be immunized.In research the 56th day, in low peptide oil-based formulation by Examination person's mean end point titre is 1.87 (4 potency having higher than detection limit in 8 subjects).It is studying the 56th day, The average log10 endpoint titers of subject in low peptide alum based formulation are 0.73, and (2 in 8 in subject have and are higher than inspection Survey the potency of limit).The average log10 endpoint titers of subject in research the 56th day, high peptide oil-based formulation are 2.08 (8 5 potency having higher than detection limit in name subject).Subject in research the 56th day, high peptide alum based formulation Average log10 endpoint titers be 0.44 (1 in the 8 subjects potency having higher than detection limit).In research the 56th The average log10 endpoint titers (collecting (pooled) from each part (arm) of research) of subject in its placebo are 0 (0 potency having higher than detection limit in 8 subjects).

The results show that these are in research the 56th day, and after single be immunized, the oil-based formulation of low dosage volume is than low dosage body Long-pending alum based formulation is significant more effective in terms of generating immune response --- contain same amount of peptide antigen.By oil-based formulation The antibody titer of induction, which continues until, at least studies the 236th day (in the group for receiving low dosage peptide) and until at least studying the 84 days (in the group for receiving high dose peptide).

Embodiment 3-RSV SHe A and RSV SHe B potency compares

6-8 week old is obtained without cause of disease from Charles River Laboratories (St.Constant QC, Canada) The female CD1 mouse of body, and disposed according to mechanism guide, wherein the air circulation in filter control is lauched and food Without limitation.

RSV SHe A peptide antigen dimer is bought from PolyPeptide Group (San Diego, USA) (NKLCEYNVFHNKTFELPRARVNT；SEQ ID NO:1) and SHe B peptide antigen monomer (NKLSEHKTFSNKTLEQGQMYQINT；SEQ ID NO:2).By adding at 37 DEG C in water/DMSO/ acetic acid mixture Heat makes RSV SHe A peptide antigen dimerization overnight.The RSV SHe A peptide that vaccine for the research contains 0.5 mcg/ml is anti- Former dimer and RSV SHe peptide antigen monomer.Not plus the oil base vaccine of adjuvant contains the oil of synthesis 1,2- bis- of 120 mg/mls Docosahexaenoyl-sn-glycero -3- phosphocholine lipid (DOPC；Lipoid, GmbH, Germany) and 12 mg/mls cholesterol (Lipoid, GmbH, Germany).Vaccine delivering in ISA 51VG oil (SEPPIC, France).The oil base vaccine of addition adjuvant also contains There is the lipopeptid adjuvant (PAM of 0.04 mg/ml₃Cys-Ser-(Lys)4；SEQ ID NO:3；PolyPeptide, San Diego, USA).The water base vaccine only peptide antigen containing 0.5 mg/ml, and buffered in 10 mMs of sodium acetate pH 7.0 It is delivered in liquid.

Mouse group (n=8) receives intramuscular inoculation twice for the 0th day and the 28th day in research, 50 microlitres of dose volumes Every kind of three kinds of vaccines.At regular intervals by mouse bloodletting to collect serum, and immune response passes through enzyme linked immunosorbent assay (ELISA) (ELISA) it is assessed to detect anti-RSV SHe A or RSV SHe B antibody.

In brief, 96 hole microtiter plates are taken the photograph with RSV SHe A or RSV SHe B antigen (1 mcg/ml) 4 It is coated with overnight, is closed under 37 degrees Celsius 30 minutes with 3% gelatin, then with the slurries (sera) of serial dilution 4 under family name's degree It is incubated overnight under degree Celsius.Then by secondary antibody (with alkaline phosphatase conjugation protein G, EMD Chemicals, Gibbstown, NJ, USA) each hole is added to 1:500 dilution continues 1 hour under 37 degrees Celsius.With containing 1 milligram/ The solution of milliliter pNPP hexahydrate (Sigma-Aldrich Chemie GmbH, Switzerland) is incubated for 60 After minute, 405 nanometers of extinctions in microtiter plate reader (ASYS Hitech GmbH, Austria) each hole of measurement are utilized Degree.Such as calculated described in Frey A et al. (Journal of Immunological Methods, 1998,221:35-41) Endpoint titers.The potency of calculating indicates highest dilution, wherein relative to from natural in the blood serum sample from immune mouseThe statistically significant increase of absorbance is observed in the blood serum sample of not immune mouse.Potency is expressed as end dilution Spend log10 value reciprocal.

Endpoint titers for RSV SHe A and RSV SHe B peptide antigen are presented in Fig. 3, and are summarised in table 2 and 3 In.

Table 2: the average +/- SEM of log10 endpoint titers

Table 3: the +/- SEM of mean end point titre

Compared with alum based formulation, the oil-based formulation induction of adjuvant is added for RSV SHe A (p < 0.01) and RSVSHe The significant higher potency of B (p < 0.001) peptide antigen.Compared with alum based formulation, not plus the induction of the oil-based formulation of adjuvant is directed to The significantly higher potency (p < 0.001) of SHe B peptide antigen.Compared with water-base preparation, not plus the induction of the oil-based formulation of adjuvant is directed to The more efficient valence of SHe A peptide antigen, but it is statistically not significant.These results prove to contain RSV SHe A or RSV SHe B The oil-based formulation of the addition adjuvant of peptide antigen can effectively induce significantly stronger antibody response --- compared with water base vaccine.These As a result also confirm oil-based formulation compared to the water base vaccine-induced more strong immune response for a variety of peptides.

The all publications and patents application of this specification reference is incorporated herein by reference, such as each independent publication Or patent application specifically and is individually indicated to be incorporated by reference into.The reference of any publication is disclosed in the applying date for it Before, it and is not necessarily to be construed as recognizing that the present invention haves no right by previous invention prior to this publication.

Although in order to which aforementioned hair has been described in detail by way of illustrating with strength in clearly understood purpose It is bright, but to those skilled in the art, introduction according to the present invention is in the essence for not departing from the appended claims In the case where mind or range, can carry out in addition certain variations and modification is obviously.

It has to be noticed that as used herein and in the appended claims, singular " one ", " one (kind) " and "the" includes plural reference, unless the context clearly indicates otherwise.Unless otherwise defined, all technologies and science used herein Term has meaning identical with the normally understood meaning of one skilled in the art of the present invention.

As being interpreted as in the element so combined in this paper specification with phrase "and/or" used in claims " any one or both " meaning, that is, exist in combination in some cases and discretely existing in other situations want Element.The multiple elements listed with "and/or" should be explained in the same manner, that is, so combine " one or more " element.In addition to by Except the element that "and/or" subordinate clause specifically confirms, other elements be may be optionally present, and no matter specifically those of confirmation element is related Or it is uncorrelated.Therefore, as non-limiting examples, when with open language for example "comprising" is used in combination when, to " A and/or B " Reference can only refer to A (optionally including the element other than B) in one embodiment；Only refer in another embodiment B (optionally includes the element other than A)；In yet another implementation, refer to both A and B (optionally other elements)； Deng.

As used in this paper specification and claims, "or" be should be read to include and "and/or" as defined above Identical meaning.For example, "or" or "and/or" should be interpreted inclusive when separating the project in table, that is, include number At least one of a element or the element of list, but also include more than one, and, optionally, add unlisted project.

As used herein, no matter in specification or the appended claims, transitional term "comprising", " comprising ", " carrying ", " having ", " containing ", " being related to " etc. should be understood it is inclusive or open (i.e., it is intended that include but is not limited to), and They are not excluded for unlisted element, material or method and step.Relative to this paper claim and illustrative embodiments section, only Transition phrase " by ... constitute " and " substantially by ... constitute " be closing or semiclosed transition phrase respectively.Transition phrase " by ... constitute " exclude not specifically enumerated any element, step or ingredient.Transition phrase " substantially by ... constitute " will Scope limitation is in specified element, material or step and does not influence substantially disclosed herein and/or claimed invention Those of essential characteristic (one or more).

Bibliography:

1)Agger,E.M.；Rosenkrands,I.；Olsen,A.W.；Hatch,G.；Williams,A.；Kritsch, C.；Lingnau,K.；von Gabain,A.；Andersen,C.S.；Korsholm,K.S.；Andersen,P.(2006) Protective immunity to tuberculosis with Ag85B-ESAT-6 in a synthetic cationic adjuvant system IC31,Vaccine24(26),5452-5460.

2)Alexopoulou,L.；Holt,A.C.；Medzhitov,R.；Flavell,R.A.(2001)Recognition of double-stranded RNA and activation of NF-_κB by Toll-like receptor 3,Nature 413(6857),732-738.

3)Altschul,S.F.；Gish,W.；Miller,W.；Myers,E.W.；Lipman,D.J.(1990)Basic local alignment search tool,J.Mol.Biol.215(3),403-410.

4)Altschul,S.F.；Madden,T.L.；Schaffer,A.A.；Zhang,J.；Zhang,Z.；Miller, W.；Lipman,D.J.(1997)Gapped BLAST and PSI-BLAST:a new generation of protein database search programs,Nucleic Acids Res 25,3389-3402

5)Anderson,R.；Huang,Y.；Langley,J.M.(2010Prospects for defined epitope vaccines for respiratory syncytial virus,Future Microbiol5(4),585-602.

6)Awasthi,A.；Mehrotra,S.；Bhakuni,V.；Dutta,G.P.；Levy,H.B.；Maheshwari, R.K.(1997)Poly ICLC enhances the antimalarial activity of chloroquine against multidrug-resistant Plasmodium yoeliinigeriensis in mice,J.Interferon Cytokine Res.17(7),419-423.

7)Banga,A.K.(1995)Therapeutic Peptides and Proteins,Formulation, Processing and Delivery Systems,Lancaster,PA:Technomic Publishing Co.

8)Bever,C.T.Jr.；Salazar,A.M.；Neely,E.；Ferraraccio,B.E.；Rose,J.W.； McFarland H.F.；Levy,H.B.；McFarlin,D.E.(1986)Preliminary trial of poly ICLC in chronic progressive multiple sclerosis,Neurology36(4),494-498.

9)Bobst,A.M.；Langemeier,P.W.；Torrence,P.F.；De Clercq,E.(1981) Interferon Induction by Poly(inosinic acid)·Poly(cytidylic acid)Segmented by Spin-Labels,Biochemistry20(16),4798-4803.

10)Brewer,K.D.；Lake,K.；Pelot,N.；Stanford,M.M.；DeBay,D.R.；Penwell,A.； Weir,G.M.；Karkada,M.；Mansour,M.；Bowen,C.V.(2014)Clearance of depot vaccine SPIO-labeled antigen and substrate visualized using MRI,Vaccine 32(51),6956- 6962.

11)Caruthers,M.H.；Beaucage,S.L.；Efcavitch,J.W.；Fisher,E.F.；Matteucci, M.D.；Stabinsky,Y.(1980)New chemical methods for synthesizing polynucleotides, Nucleic Acids Res.Symp.Ser.(7),215-223.

12)Celis,E.；Ou,D.；Otvos,L.Jr.(1988)Recognition of hepatitis B surface antigen by human T lymphocytes.Proliferative and cytotoxic responses to a major antigenic determinant defined by synthetic peptides,J.Immunol.140,1808- 1815.

13)Chanock,R；Finberg L.(1957)Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent(CCA).II.Epidemiologic aspects of infection in infants and young children,Am J Hyg.66(3),291-300.

14)Chanock,R.；Roizman B.；Myers,R.(2008)Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent(CCA) .II.Epidemiologic aspects of infection in infants and young children,Am J Hyg.66(3),281-90.

15)Chirigos,M.A.；Schlick,E.；Ruffmann,R.；Budzynski,W.；Sinibaldi,P.； Gruys,E.(1985)Pharmacokinetic and therapeutic activity of polyinosinic- polycytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(I,C)-LC],J.Biol.Response Mod.4(6),621-627.

16)Chong,P.；Zobrist,G.；Sia,C.；Loosmore,S.；Klein,M.(1992) Identification of T-and B-cell epitopes of the S2 and S3 subunits of pertussis toxin by use of synthetic peptides,Infect Immun.60,4640–4647.

17)Collins,P.L.；Olmsted,R.A.；Johnson,P.R.(1990)The small hydrophobic protein of human respiratory syncytial virus:comparison between antigenic subgroups A and B,J Gen Virol.71(7),1571-6.

18)Cui,Z.；Qiu,F.(2006)Synthetic double-stranded RNA poly(I:C)as a potent peptide vaccine adjuvant:therapeutic activity against human cervical cancer in a rodent model,Cancer Immunol.Immunother.55(10),1267-1279.

19)deClercq,E.；Hattori,M.；Ikehara,M.(1975)Antiviral activity of polynucleotides:copolymers of inosinic acid and N2-dimethylguanylic of 2- methylthioinosinic acid,Nucleic Acids Res.2(1),121-129.

20)de Clercq,E.；Torrence,P.F.；Stollar,B.D.；Hobbs,J.；Fukui,T.； Kakiuchi,N.；Ikehara,M.(1978)Interferon induction by a 2'-modified double- helical RNA,poly(2'-azido-2'-deoxyinosinic acid).polycytidylic acid, Eur.J.Biochem.88(2),341-349.

21)Demotz,S.；Lanzavecchia,A.；Eisel,U.；Niemann,H.；Widmann,C.；Corradin, G.P.(1989)Delineation of several DR-restricted epitopes in tetanus toxin, J.Immunol.142,394-402.

22)Diethelm-Okita,B.M.；Okita,D.K.；Banaszak,L.；Conti-Fine,B.M.(2000) Universal epitopes for human CD4+cells on tetanus and diphtheria toxins, J.Infect.Dis.181,1001–1009.

23)Dong,L.W.；Kong,X.N.；Yan,H.X.；Yu,L.X.；Chen,L.；Yang,W.；Liu,Q.；Huang, D.D.；Wu,M.C.；Wang,H.Y.(2008)Signal regulatory protein alpha negatively regulates both TLR3 and cytoplasmic pathways in type I interferon induction, Mol.Immunol.45(11),3025-3035.

24)Droller,M.J.(1987)Immunotherapy of metastatic renal cell carcinoma with polyinosinic-polycytidylic acid,J.Urol.137(2),202-206.

25)Durie,B.G.；Levy,H.B.；Voakes,J.；Jett,J.R.；Levine,A.S.(1985)Poly(I, C)-LC as an interferon inducer in refractory multiple myeloma,J.Biol.Response Mod.4(5),518-524.

26)Frezard,F.(1999)Liposomes:from biophysics to the design of peptide vaccines,Braz.J.Med.Bio.Res.32,181-189.

27)Frey,A.；Di Canzio,J.；Zurakowski,D.(1998)A statistically defined endpoint titer determination method for immunoassays,Journal of Immunological Methods 221:35-41.

28)Fujimura,T.；Nakagawa,S.；Ohtani,T.；Ito,Y.；Aiba,S.(2006)Inhibitory effect of the polyinosinic-polycytidylic acid/cationic liposome on the progression of murine B16F10melanoma,Eur.J.Immunol.36(12),3371-3380.

29)Fukui,T.；Kakiuchi,N.；Ikehara,M.(1977)Polynucleotides.XLV Synthesis and properties of poly(2'-azido-2'-deoxyinosinic acid),Nucleic Acids Res.4 (8),2629-2639.

30)Gowen,B.B.；Wong,M.H.；Jung,K.H.；Sanders,A.B.；Mitchell,W.M.； Alexopoulou,L.；Flavell,R.A.；Sidwell,R.W.(2007)TLR3 is essential for the induction of protective immunity against Punta Toro Virus infection by the double-stranded RNA(dsRNA),poly(I:C12U),but not Poly(I:C):differential recognition of synthetic dsRNA molecules,J.Immunol.178(8),5200-5208.

31)Gregoriadis G.(1990)Immunological adjuvants:a role for liposomes, Immunol.Today 11,89-97.

32)Greene,J.J.；Alderfer,J.L.；Tazawa,I.；Tazawa,S.；Ts'o,P.O.；O'Malley, J.A.；Carter,W.A.(1978)Interferon induction and its dependence on the primary and secondary structure of poly(inosinic acid).poly(cytidylic acid), Biochemistry 17(20),4214-4220.

33)Guschlbauer,W.；Blandin,M.；Drocourt,J.L.；Thang,M.N.(1977)Poly-2'- deoxy-2'-fluoro-cytidylic acid:enzymatic synthesis,spectroscopic characterization and interaction with poly-inosinic acid,Nucleic Acids Res.4 (6),1933-1943.

34)Hendrix,C.W.；Margolick,J.B.；Petty,B.G.；Markham,R.B.；Nerhood,L.； Farzadegan,H.；Ts'o,P.O.；Lietman,P.S.(1993)Biologic effects after a single dose of poly(I):poly(C12U)in healthy volunteers,Antimicrob.Agents Chemother.37(3),429-435.

35)Horn,T.；Vasser,M.P.；Struble,M.E.；Crea,R.(1980)Synthesis of oligonucleotides on cellulose.Part II:Design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for gastric inhibitory polypeptide(GIP),Nucleic Acids Symp.Ser.(7),225-232.

36)Houston,W.E.；Crabbs,C.L.；Stephen,E.L.；Levy,H.B.(1976)Modified polyriboinosinic-polyribocytidylicacid,an immunological adjuvant, Infect.Immun.14(1),318-319.

37)Ichinohe,T.；Tamura,S.；Kawaguchi,A.；Ninomiya,A.；Imai,M.；Itamura,S.； Odagiri,T.；Tashiro,M.；Takahashi,H.；Sawa,H.；Mitchell,W.M.；Strayer,D.R.；Carter, W.A.；Chiba,J.；Kurata,T.；Sata,T.；Hasegawa,H.(2007)Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine,J.Infect.Dis.196(9),1313- 1320.

38)Johnston,M.I.；Stollar,B.D.；Torrence,P.F.；Witkop,B.(1975)Structural features of double-stranded polyribonucleotides required for immunological specificity and interferon induction,Proc.Natl.Acad.Sci.U.S.A.72(11),4564- 4568.

39)Kamath,A.T.；Valenti,M.P.；Rochat,A.F.；Agger,E.M.；Lingnau,K.；von Gabain,A.；Andersen,P.；Lambert,P.H.；Siegrist,C.A.(2008)Protective anti- mycobacterial T cell responses through exquisite in vivo activation of vaccine-targeted dendritic cells,Eur.J.Immunol.38(5),1247-1256.

40)Karron RA,Respiratory syncytial virus and parainfluenza virus vaccines,in Vaccines,Plotkin S.A.；Orenstein W.A.；and Offit,P.A.,Editors.2013, Elsevier Saunders,1146-1153.

41)Kawaoka,Y.；Yamnikova,S.；Chambers,T.M.；Lvov,D.K.；Webster,R.G.(1990) Molecular characterization of a new hemagglutinin,subtype H14,of influenza A virus,Virology179(2),759-767

42)Kende,M.；Lupton,H.W.；Rill,W.L.；Gibbs,P.；Levy,H.B.；Canonico,P.G. (1987)Ranking of Prophylactic Efficacy of Poly(ICLC)against Rift Valley Fever Virus Infection in Mice by Incremental Relative Risk of Death, Antimicrob.Agents Chemother.31(8),1194-1198.

43)Khanna,N.；Widmer,A.F.；Decker,M.；Steffen,I.,Halter,J.；Heim,D.； Weisser,M.；Gratwohl,A.；Fluckiger,U.；Hirsch,H.H.(2008)Respiratory syncytial virus infection in patients with hematological diseases:single-center study and review of the literature,Clin Infect Dis.46(3),402-12.

44)Kreuter,J.,ed.(1994),Colloidal Drug Delivery Systems,Vol.66,Marcel Dekker,Inc.

45)Krown,S.E.；Kerr,D.；Stewart,W.E.2nd；Field,A.K.；Oettgen,H.F.(1985) Phase I trials of poly(I,C)complexes in advanced cancer,J.Biol.Response Mod.4 (6),640-649.

46)Langley,J.M.；LeBlanc,J.C.；Smith,B.；Wang,E.E.(2003)Increasing incidence of hospitalization for bronchiolitis among Canadian children,1980- 2000,J Infect Dis.188(11),1764-7.

47)Levy,H.B；Lvovsky,E.(1978)Topical treatment of vaccinia virus infection with an interferon inducer in rabbits,J.Infect.Dis.137(1),78-81.

48)Levy,H.B.(1985)Historical overview of the use of polynucleotides in cancer,J.Biol.Response Mod.4(5),475-480.

49)Llopiz,D.；Dotor,J.；Zabaleta,A.；Lasarte,J.J.；Prieto,J.；Borrás- Cuesta,F.；Sarobe,P.(2008)Combined immunization with adjuvant molecules poly (I:C)and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects,Cancer Immunol.Immunother.57(1),19-29.

50)Merrifield,B.(1997)Concept and early development of solid-phase peptide synthesis,Methods Enzymol.289,3-13.

51)Moghaddam,A.；Olszewska,W.；Wang,B.；Tregoning,J.S.；Helson,R.； Sattentau,Q.J.；Openshaw,P.J.(2006)A potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines,Nat Med.12(8),905-7.

52)Moyle,P.M.；Toth.I.(2008)Self-adjuvantinglipopeptide vaccines, Curr.Med.Chem.15(5),506-516.

53)Nair,H.；Nokes,D.J.；Gessner,B.D.；Dherani,M.；Madhi,S.A.；Singleton, R.J.et al.,(2010)Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children:a systematic review and meta- analysis,Lancet375(9725),1545-55.

54)Nair,H.；Verma,V.R.；Theodoratou,E.；Zgaga,L.；Huda,T.；E.A.F.； Wright,P.F.；Rudan,I.；Campbell,H.(2011)An evaluation of the emerging interventions against Respiratory Syncytial Virus(RSV)-associated acute lower respiratory infections in children,BMC Public Health11 Suppl 3,S30.

55)Nakamura,O.；Shitara,N.；Matsutani,M.；Takakura,K.；Machida,H.(1982) Phase I-II trials of poly(ICLC)in malignant brain tumor patients, J.Interferon.Res.2(1),1-4.

56)Needleman,S.B.；Wunsch,C.D.(1970)A general method applicable to the search for similarities in the amino acid sequence of two proteins, J.Mol.Biol.48(3),443-453.

57)Padalko,E.；Nuyens,D.；De Palma,A.；Verbeken,E.；Aerts,J.L.；De Clercq, E.；Carmeliet,P.；Neyts,J.(2004)The Interferon Inducer Ampligen[poly(I)-poly (C₁₂U)]Markedly Protects Mice against Coxsackie B3 Virus-Induced Myocarditis, Antimicrob.Agents Chemother.48(1),267-274.

58)Pearson,W.R.；Lipman,D.J.(1988)Improved tools for biological sequence comparison,Proc.Natl.Acad.Sci.U.S.A.85(8),2444-2448.

59)Poast,J.；Seidel,H.M.；Hendricks,M.D.；Haslam,J.A.；Levy,H.B.；Baron,S. (2002)Poly I:CLC induction of the interferon system in mice:an initial study of four detection methods,J.Interferon Cytokine Res.22(10),1035-1040.

60)Puri,S.K.；Dutta,G.P.；Levy,H.B.；Maheshwari,R.K.(1996)Poly ICLC inhibits Plasmodium cynomolgi B malaria infection in rhesus monkeys, J.Interferon.Cytokine Res.16(1),49-52.

61)Remington’s Pharmaceutical Sciences,Mack Publishing Company, Easton,Pa.,USA1985

62)Riedl,K.；Riedl,R.；von Gabain,A.；Nagy,E.；Lingnau,K.(2008)The novel adjuvant strongly improves influenza vaccine-specific cellular and humoral immune responses in young adult and aged mice,Vaccine 26(27-28),3461- 3468.

63)Roberge,J.Y.；Beebe,X.；Danishefsky,S.J.(1995)A strategy for a convergent synthesis of N-linked glycopeptides on a solid support,Science 269 (5221),202-204.

64)Salazar,A.M.；Levy,H.B.；Ondra,S.；Kende,M.；Scherokman,B.；Brown,D.； Mena,H.；Martin,N.；Schwab,K.；Donovan,D.；Dougherty,D.；Pulliam,M.；Ippolito,M.； Graves,M.；Brown,H.；Ommaya,A.(1996)Long-term treatment of malignant gliomas with intramuscularly administered polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose:an open pilot study,Neurosurgery 38(6),1096-1103,discussion at 1103-1104.

65)Salem,M.L.；Kadima,A.N.；Cole,D.J.；Gillanders,W.E.(2005)Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity,J.Immunother.28(3),220-228.

66)Salem,M.L.；El-Naggar,S.A.；Kadima,A.；Gillanders,W.E.；Cole,D.J. (2006)The adjuvant effects of the toll-like receptor 3 ligand polyinosinic- cytidylic acid poly(I:C)on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu,Vaccine 24(24),5119-5132.

67)Sarma,P.S.；Shiu,G.；Neubauer,R.H.；Baron,S.；Huebner,R.J.(1969)Virus- induced sarcoma of mice:inhibition by a synthetic polyribonucleotide complex, Proc.Natl.Acad.Sci.U.S.A.62(4),1046-1051.

68)Schellack,C.；Prinz,K.；Egyed,A.；Fritz,J.H.；Wittmann,B.；Ginzler,M.； Swatosch,G.；Zauner,W.；Kast,C.；Akira,S.；von Gabain,A.；Buschle,M.；Lingnau,K. (2006)IC31,a novel adjuvant signaling via TLR9,induces potent cellular and humoral immune responses,Vaccine24(26),5461-5472.

69)Sharon,J；Rynkiewicz M.J.；Lu,Z.；Yang,C.Y.(2014)Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics,Immunology142(1),1-23.

70)Slingluff,C.L.Jr.；Yamshchikov,G.；Neese,P.；Galavotti,H.；Eastham,S.； Engelhard,V.H.；Kittlesen,D.；Deacon,D.；Hibbitts,S.；Grosh,W.W.；Petroni,G.； Cohen,R.；Wiernasz,C.；Patterson,J.W.；Conway,B.P.；Ross,W.G.(2001)Phase I trial of a melanoma vaccine with gp100(280–288)peptide and tetanus helper peptide in adjuvant:Immunologic and clinical outcomes,Clin Cancer Res.7,3012–3024.

71)Sloat,B.R.；Shaker,D.S.；Le,U.M.；Cui,Z.(2008)Nasal immunization with the mixture of PA63,LF,and a PGA conjugate induced strong antibody responses against all three antigens,FEMS Immunol.Med.Microbiol.52(2),169-179.

72)Smith,T.F.；Waterman,M.S.(1981)Comparison of biosequences, Adv.Appl.Math.2(4),482-489.

73)So,N.S.Y.；Ostrowski,M.A.；Gray-Owen,S.D.(2012)Vigorous Response of Human Innate Functioning IgM Memory B Cells upon Infection by Neisseria gonorrhoeae,J.Immunol.188(8),4008-4022.

74)Stephen,E.L.；Sammons,M.L.；Pannier,W.L.；Baron,S.；Spertzel,R.O.； Levy,H.B.(1977)Effect of a nuclease-resistant derivative of polyriboinosinic- polyribocytidylic acid complex on yellow fever in rhesus monkeys (Macacamulatta),J.Infect.Dis.136(1),122-126.

75)Talmadge,J.E.；Adams,J.；Phillips,H.；Collins,M.；Lenz,B.；Schneider, M.；Chirigos,M.(1985)Immunotherapeutic potential in murine tumor models of polyinosinic-polycytidylic acid and poly-L-lysine solubilized by carboxymethylcellulose,Cancer Res.45(3),1066-1072.

76)The United States Pharmacopoeia:The National Formulary(USP 24 NF19)published in 1999

77)Theriault,R.L.；Hortobagyi,G.N.；Buzdar,A.U.；Levy,H.B.；Hersh,E.M. (1986)Evaluation of polyinosinic-polycytidylic and poly-L-lysine in metastatic breast cancer,Cancer Treat.Rep.70(11),1341-1342.

78)Trumpfheller,C.；Caskey,M.；Nchinda,G.；Longhi,M.P.；Mizenina,O.； Huang,Y.；Schlesinger.S.J.；Colonna,M.；Steinman,R.M.(2008)The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine,Proc.Natl.Acad.Sci.U.S.A.105(7),2574-2579.

79) Van Regenmortel, M.H. (2009) What is a B-cell epitope?, Methods Mol Biol.524,3-20.

80)Walsh,E.E.；Falsey,A.R.(2004)Humoral and mucosal immunity in protection from natural respiratory syncytial virus infection in adults,J Infect Dis.190(2),373-8.

81)Webster,R.G.；Laver,W.G.；Air,G.M.Antigenic variation among type A influenza viruses,p.127-168.In:Palese,P.&Kingsbury,D.W.,eds.Genetics of influenza viruses.(New York:Springer-Verlag,1983).

82)Zaks,K.；Jordan,M.；Guth,A.；Sellins,K.；Kedl,R.；Izzo,A.；Bosio,C.；Dow, S.(2006)Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9agonists complexed to cationic liposomes,J.Immunol.176 (12),7335-7345.

83)Yoneyama,M.；Kikuchi,M.；Natsukawa,T.；Shinobu,N.；Imaizumi,T.； Miyagishi,M.；Taira,K.；Akira,S.；Fujita,T.(2004)The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses, Nat.Immunol.5(7),730-737.

84)Kametani,Y.；Miyamoto,A.；Tsuda,B.；Tokuda,Y.(2015)B Cell Epitope- Based Vaccination Therapy,Antibodies 4(3),225-239.

85)Zhu,X.；Nishimura,F.；Sasaki,K.；Fujita,M.；Dusak,J.E.；Eguchi,J.； Fellows-Mayle,W.；Storkus,W.J.；Walker,P.R.；Salazar,A.M.；Okada,H.(2007)Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models,J.Transl.Med.5(10),1-15.

Sequence table

<110>Immunovaccine Technologies Inc.

<120>using low dosage volume B cell epitope composition in the method for the induction of antibodies immune response in the experimenter

<130> 78961-181

<140> PCT/CA2016/xxxxxx

<141> 2016-09-27

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 23

<212> PRT

<213>artificial sequence

<220>

<223>RSV SHe A peptide

<400> 1

Asn Lys Leu Cys Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu

1 5 10 15

Pro Arg Ala Arg Val Asn Thr

20

<210> 2

<211> 24

<212> PRT

<213>artificial sequence

<220>

<223>RSV SHe B peptide

<400> 2

Asn Lys Leu Ser Glu His Lys Thr Phe Cys Asn Lys Thr Leu Glu Gln

1 5 10 15

Gly Gln Met Tyr Gln Ile Asn Thr

20

<210> 3

<211> 6

<212> PRT

<213>artificial sequence

<220>

<223>palmitinic acid adjuvant

<220>

<221> Misc_Feature

<222> (1)..(1)

<223>it is connected to PAM2 or PAM3

<400> 3

Cys Ser Lys Lys Lys Lys

1 5

<210> 4

<211> 64

<212> PRT

<213>artificial sequence

<220>

<223>people RSV SH A subgroup

<400> 4

Met Glu Asn Thr Ser Ile Thr Ile Glu Phe Ser Ser Lys Phe Trp Pro

1 5 10 15

Tyr Phe Thr Leu Ile His Met Ile Thr Thr Ile Ile Ser Leu Leu Ile

20 25 30

Ile Ile Ser Ile Met Ile Ala Ile Leu Asn Lys Leu Cys Glu Tyr Asn

35 40 45

Val Phe His Asn Lys Thr Phe Glu Leu Pro Arg Ala Arg Val Asn Thr

50 55 60

<210> 5

<211> 65

<212> PRT

<213>artificial sequence

<220>

<223>people RSV SH B subgroup

<400> 5

Met Gly Asn Thr Ser Ile Thr Ile Glu Phe Thr Ser Lys Phe Trp Pro

1 5 10 15

Tyr Phe Thr Leu Ile His Met Ile Leu Thr Leu Ile Ser Leu Leu Ile

20 25 30

Ile Ile Thr Ile Met Ile Ala Ile Leu Asn Lys Leu Ser Glu His Lys

35 40 45

Thr Phe Cys Asn Lys Thr Leu Glu Gln Gly Gln Met Tyr Gln Ile Asn

50 55 60

Thr

65

<210> 6

<211> 23

<212> PRT

<213>artificial sequence

<220>

<223>RSV SHe A peptide variant

<400> 6

Asn Lys Leu Ser Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu

1 5 10 15

Pro Arg Ala Arg Val Asn Thr

20

<210> 7

<211> 9

<212> PRT

<213>artificial sequence

<220>

<223>HPV R9F peptide

<400> 7

Arg Ala His Tyr Asn Ile Val Thr Phe

1 5

<210> 8

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>CpG ODN

<400> 8

tccatgacgt tcctgacgtt 20

<210> 9

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>PolyI:C polynucleotides

<220>

<221>modify _ base

<222> (1)..(1)

<223>inosine

<220>

<221>modify _ base

<222> (3)..(3)

<223>inosine

<220>

<221>modify _ base

<222> (5)..(5)

<223>inosine

<220>

<221>modify _ base

<222> (7)..(7)

<223>inosine

<220>

<221>modify _ base

<222> (9)..(9)

<223>inosine

<220>

<221>modify _ base

<222> (11)..(11)

<223>inosine

<220>

<221>modify _ base

<222> (13)..(13)

<223>inosine

<220>

<221>modify _ base

<222> (15)..(15)

<223>inosine

<220>

<221>modify _ base

<222> (17)..(17)

<223>inosine

<220>

<221>modify _ base

<222> (19)..(19)

<223>inosine

<220>

<221>modify _ base

<222> (21)..(21)

<223>inosine

<220>

<221>modify _ base

<222> (23)..(23)

<223>inosine

<220>

<221>modify _ base

<222> (25)..(25)

<223>inosine

<400> 9

ncncncncnc ncncncncnc ncncnc 26

<210> 10

<211> 16

<212> PRT

<213>artificial sequence

<220>

<223>tetanus toxin peptide A16L

<400> 10

Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210> 11

<211> 13

<212> PRT

<213>artificial sequence

<220>

<223>T assists epitope PADRE

<220>

<221>MISC_ feature

<222> (3)..(3)

<223>Xaa can be cyclohexyl alanyl

<400> 11

Ala Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Ala

1 5 10

<210> 12

<211> 21

<212> PRT

<213>artificial sequence

<220>

<223>tetanus toxoid peptide F21E

<400> 12

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

Claims

1. for the method for the induction of antibodies immune response in the experimenter, including parenterally giving low dosage to the experimenter The composition of volume, the composition include:

It include the antigen of B cell epitope；

Amphipathic compound；And

Hydrophobic carrier,

Wherein the low dosage volume of the composition is less than 100 μ l, and induction is thin for the B in the experimenter The antibody mediated immunity response of born of the same parents' epitope.

2. method described in claim 1, wherein the low dosage volume is about 50 μ l, about 60 μ l, about 70 μ l, about 80 μ l or about 90μl。

3. method of any of claims 1 or 2, wherein the low dosage volume is 50 μ l.

4. the method described in any one of claims 1 to 3 is given only the single of the composition including (i) and gives, or (ii) Single give for the first time for being given only the composition is given with single reinforcement.

5. method as claimed in claim 4, in which:

It is described it is single for the first time give after about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 Week, about 10 weeks, about 11 weeks, about 12 weeks or longer time, the Xiang Suoshu experimenter provide the single reinforcement and give；Or

It is described it is single for the first time give after about 56 days, the Xiang Suoshu experimenter provide it is described it is single reinforcement give.

6. method described in any one of claims 1 to 5, wherein the composition of the low dosage volume:

It is answered at least as early as 28 days induction detectable antibody mediated immunities in the experimenter after giving the composition for the first time It answers；

Induction of antibodies immune response, the antibody mediated immunity response after giving the composition in the experimenter for the first time Continue at least 84 days or at least 236 days；

The 56th day, the 84th day or the 236th day after giving, at least 75%, at least 80%, at least 85%, at least 90%, extremely Induction of antibodies immune response in few 95% or at least 100% experimenter；

The 84th day or the 236th day at least after giving, the induction of antibodies immune response in the 100% treated experimenter； And/or

It induces enhanced compared to the antibody mediated immunity response induced in the experimenter for giving the antigen that alum composition is prepared Antibody mediated immunity response, wherein the alum composition include the antigen, alum adjuvant, mineral adjuvant (such as ) and aqueous carrier Alhyrogel.

7. method of claim 6, wherein the composition induction of the low dosage volume compared to give it is described The enhanced antibody mediated immunity response of the antibody mediated immunity response induced in the experimenter of alum composition, in which:

Each in the composition and the alum composition includes the antigen of 10 μ g, and in the composition In the case where, mean antigen specific antibody endpoint titers: (i) the 28th day up at least about than the alum composition after giving 123.2 again；And/or (ii) at the 56th day than up at least about 16.7 times of the alum composition；

Each in the composition and the alum composition includes the antigen and the composition of 25 μ g Mean antigen specific antibody endpoint titers: (i) the 28th day after giving, than up at least about 35.3 times of the alum composition； And/or (ii) at the 56th day, than up at least about 13.1 times of the alum composition；

Each in the composition and the alum composition includes the antigen of 10 μ g, and the 56th after giving It, is when being treated with the composition, the percentage ratio of the experimenter with detectable antigen-specific antibodies immune response With up at least about 2.0 times of the alum composition；

Each in the composition and the alum composition includes the antigen of 25 μ g, and the 56th after giving It, is when being treated with the composition, the percentage ratio of the experimenter with detectable antigen-specific antibodies immune response With up at least about 5.0 times of the alum composition；And/or

About the whole body adverse events of overall demand and/or the adverse events of overall non-demand, the composition has acceptable Safety.

8. method described in any one of claims 1 to 7, wherein the experimenter is 0-2 years old, 50-64 years old or over-65s Age.

9. method described in any item of the claim 1 to 8, wherein the composition further includes adjuvant, the adjuvant choosing From PolyI:C polynucleotides adjuvant, the adjuvant based on lipid, lipid A mimic or its analog, or any combination thereof.

10. method described in any one of claims 1 to 9, wherein the antigen is: length is the peptide of 5 to 50 amino acid The polynucleotides of antigen or the coding peptide antigen；Or the synthetic peptide in the experimenter with natural weak immunogene is anti- It is former.

11. method described in any one of claims 1 to 10, wherein the antigen includes respiratory syncytial virus (RSV) (RSV) Small hydrophobin extracellular domain (SHe) or its segment, or the small hydrophobin by respiratory syncytial virus (RSV) (RSV) Extracellular domain (SHe) or its segment constitute.

12. method described in claim 11, wherein the SHe is derived from RSV plants of RSV plants of A subgroup people or B subgroup people.

13. method described in any one of claims 1 to 12, wherein the antigen includes amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO1), with SEQ ID NO:1 at least 75% homologous amino acid sequence or The segment of SEQ ID NO:1, or by amino acid sequence NKLCEYNVFHNKTFELPRARVNT (SEQ ID NO1) and SEQ The segment of the homologous amino acid sequence of ID NO:1 at least 75% or SEQ ID NO:1 are constituted.

14. method described in any one of claims 1 to 13, wherein the amphipathic compound is lipid or lipid mixture； And/or the hydrophobic carrier is the mixture of oil or oil.

15. method described in any one of claims 1 to 14, wherein the composition includes:

Short synthesis lipopeptid, is PAM₃Cys-Ser-(Lys)4(SEQ ID NO:3)；And

-ISA 51 VG。

16. method described in any one of claims 1 to 15, wherein the composition is not aqueous or generally not aqueous.

17. method described in any one of claims 1 to 16, including giving the low dosage volume by intramuscular injection The composition.

18. method described in any one of claims 1 to 17 is used to treat or prevent the infectious disease in the experimenter Disease.

19. the composition of low dosage volume is directed to the application of the antibody mediated immunity response of B cell epitope for inducing in the experimenter, The composition includes:

It include the antigen of B cell epitope；

Amphipathic compound；And

Hydrophobic carrier,

Wherein the composition is given for parenteral and the low dosage volume of the composition is less than 100 μ l.

20. for according to claim 1 to the compositions or agents box of method described in any one of 18.